Antibacterial composition containing (-) (cis-1 2-epoxypropyl) phosphoric acid

ABSTRACT

(-) (CIS- 1,2-EPOXYPROPYL)-PHOSPHORIC ACID, A WATER SOLUBLE ACIDIC SUBSTANCE, AND ITS SALTS ARE ACTIVE AGAINST BOTH GRAM-POSITIVE AND GRAM-NEGATIVE BACTERIA. THE ANTIBIOTIC IS PRODUCED BY GROWING NEWLY-FOUND AND HITHERTO UNDESCRIBED STRAINS OF STREPTOMYCES ON SUITABLE FERMENTATION MEDIUMS.

United States Patent 3,639,590 ANTIBACTERIAL COMPOSITION CONTAINING (ClS-LZ-EPOXYPROPYL) PHOSPHORIC ACID David Hendlin, Springfield, and Edward O. Stapley, Spotswood, N.J., and Sagrario Mochales del Va] and .lusto Martinez Mata, Madrid, Spain, assignors to Merck & (10., Inc., Rahway, NJ.

N 0 Drawing. Continuation-impart of applications Ser. No. 655,757, July 25, 1967, and Ser. No. 679,165, Oct. 30, 1967. This application May 9, 1968, Ser. No. 728,059

Int. Cl. A611: 21/00 US. Cl. 424203 9 Claims ABSTRACT OF THE DISCLOSURE (Cis-1,2-epoxypropyl)-phosphonic acid, a Water soluble acidic substance, and its salts are active against both gram-positive and gram-negative bacteria. The antibiotic is produced by growing newly-found and hitherto undescribed strains of Streptomyces on suitable fermentation mediums.

CROSS REFERENCE TO RELATED APPLICATIONS This is a continuation-in-part of copending applications, Ser. No. 655,757 filed July 25, 1967, now abandoned and Ser. No. 679,165, filed Oct. 30, 1967, now abandoned.

BACKGROUND OF THE INVENTION The discovery of the remarkable antibiotic properties of penicillin stimulated great interest in this field which has resulted in the finding of many other valuable antibiotic substances such as: streptomycin, gramicidin, subtilin, bacitracin, chlortetracycline, oxytetracycline and the like. In general, such antibiotics are particularly active against certain gram-negative bacteria, others are active against gram-positive bacteria, and some are active against both gram-negative and gram-positive bacteria. However, the activity of these known antibiotics is usually limited to a few pathogenic microorganisms, and work has been continued in this field in an attempt to find other antibiotics which would be effective against other pathogens.

Although some of these antibiotics have been found to be invaluable in the treatment of various diseases, it is found that certain strains of some pathogens develop a resistance to a particular antibiotic, and as a result, the antibiotic is no longer active against such resistant strains.

Accordingly, the deficiencies of the known antibiotics have stimulated further research to find other antibiotics whinch will be active against a wider range of pathogens as Well as resistant strains of particular microorganisms.

SUMMARY OF THE INVENTION This invention relates to new antibiotic agents and processes of preparing the same. More particularly, it is concerned with a new antibiotic substance, herein called Antibiotic 833A or by its chemical name (cis-l,2-epoxypropyl)-phosphonic acid, salts thereof and with processes for their production.

It is an object of the present invention to provide a new and useful antibiotic which is highly effective in inhibiting the growth of various gram-negative and grampos-itive microorgansms. Another object is to provide a process for the preparation of this novel antiobiotic substance-by the fermentation of nutrient mediums with suitable strains ofheretofore undescribed microorganisms. Other objects will be apparent from the detailed description of this invention 'hereinafter'provided.

The new antibiotic substance of the present invention is formed. by growing, under controlled conditions,

previously-unknown strains of microorganisms. Thus, the new antibiotic is produced by growing Antiobiotic 833A producing strains of the genus Streptomyces. One such microorganism, which was isolated from soil, has been designated MA-2898 in the culture collection of Merck & Co., !Inc., Rahway, NJ. The original isolate of the Antibiotic 833A-producing culture, obtained as a single colony from soil, was plated onto an agar plate of the following composition:

G. liter Yeast extract 10 Glucose 10 Agar 20 MgSO .7H O 0.05

Phospate buffer, 2 ml. Distilled water q.s., balance.

1 91 g. KHQPOi and 95.0 g. NazHPOi made up to 1 liter with distilled water.

After several days of growth, it was found that several colony types were produced on the agar plate, namely, both white and pink sporulating types. Single colony subisolates were found to vary significantly in pigmentation, aerial mycelium, extent of sporulation, and fermentation of carbohydrates. However, each of the isolates or variants produced (cis-1,2-epoxypropyl)-phosphonic acid. Subisolatess of the parent microorganism are identified as MA-2911, MA-2912, and MA2913 in the previouslymentioned Merck culture collection. The parent culture was also found to degrade to a non-sporulating form with significant frequency, which does not produce the new antibiotic of this invention.

The parent culture, MA2898, and the subisolates MA2911, MA-2912, and MA2913 have been deposited in the culture collection of the American Type Culture Collection where they are available as ATCC 21096, ATCC 21097, ATCC 21098, and ATCC 21099, respectively. The same cultures have also been placed on permanent deposit with the culture collection of the Northern Utilization Research and Development Branch of the US. Department of Agriculture at Peoria, 111., and have been assigned the culture numbers NRRL B-3357, NRRL B-3358, N RRL B3359, and NRRL B-3360, respectively.

Based upon extensive taxonomic studies, these microorganisms have been classified in the species Streptomyces fraa'iae. It is a unique strain of the species, differing in one or more characteristics, from all descriptions of members of the species found in standard reference works.

For the taxonomic studies, the parent culture and the single colony isolates were stabilized by the process of lyophilization of spore stocks to insure reproducible results. Cultures used for examination of morphological and physiological characteristics were grown from these stable stocks. Certain of the single colony isolates differed suificiently from the standard description of S. fradiae to the extent that, standing alone, they might be considered as new species. The fact that all single colony isolates originated from a single stock and the fact that the parent culture shows a high order of variability justified including the culture and its variants in the widest scope of their characteristics, as strains of S. fradiae. Waksman has defined S. fradiae broadly (The Actinomycetes, volume II, p. 212) as follows: The characteristics of the species are that it is non-chromogenic, strongly proteolytic, and produces the characteristic seashell pink aerial mycelium on various synthetic media; on organic media, orange color growth is produced without any aerial mycelium. The above-mentioned strains of the microorganism producing Antibiotic 833A fit this mycelium is often observed on organic media.

The characteristics of the parent isolate and of four single colony isolates are shown in Table 1 in comparison with the description of the type species Streptomyces fradiae as it appears in Bergeys Manual of Determinative Bacteriology, 7th edition, pp. 799-800. All of the readings shown in the table were taken after three weeks TABLE 1 [Characteristics of antibiotic 833A producing strains and Streptomyces fradiae] Original Culture Single-colony isolates derived from culture MA-2898 MA-2898 (ATCC 21096) MA-29l1 (ATCC 21097) MA-2912 (ATCC 21098) MA-2913 (ATCC 21099) Streptomyces fradiae Morphology Sporophores are long, branched, straight or fiexuous. Spores are oval to rod-shaped, 0.9 by 1.2-1.7

(applies to all microns, in chains of more than 10. (070K) four MA numbers). 244

Observed on tomato paste-oatmeal agar, glycerol-asparaginase agar, egg albumen agar, and Czapek-Dox agar.

Aerial mycelium Straight, branching filaments and hyphae; No spirals. Conidia rod-shaped or ellipsoidal, 0.5 by 0.7-1.25 microns.

Tomato Paste- Reverse-orange. Reverseorange. Reversedeep orange. Reverse-deep orange. oatmeal agar. Aerial mycelium-flat, Aerial mycelium-fiat, Aerial mycelium-flat, Aerial mycelium-flat,

powdery, mixture of powdery, pinkishpowdery cinnamonpowdery, dustypink and cream cream (3 ca). pink (approx. 5 go). pink (approx. 5 cc).

c Soluble pigmenttan. Soluble pigment-tan. Soluble pigment-tan. Spreading fan-shaped areas of cream to tan vegetative growth with no aerial mycelium extending from main mass of growth. Soluble pigment-tan.

Nutrient agar Reverse-dark cream Reversecream. Reversecrcarn to Reverse-cream.

to yellow. Aerial myeelium-dlat, y w. Vegetativecream- Aerial mycelium-flat, powdery, ivory. Aerial mycelium--ilat, colored, spreading. powdery, pale flesh No soluble pigment. powdery, pale flesh Aerial mycelium pi p k. moderate, grayish- No soluble pigment. No soluble pigment. white.

No soluble pigment.

Agar Yellowish growth,

becoming orangeyellow, restricted.

No soluble pigment.

Egg albumen agar Reverse-white to Reverse-white to Reverse-white to Reverseeream to ye ow. y W. yellow. pinkish yellow. Aerial myceliu mflat, Aerial mycelium-flat, Aerial myceliumfiat, Spreading growth.

powdery, ivory with powdery, cream powdery, mottled Aerial mycelium-flat, pink tint, very pale shading into very pink, cream and powdery, cream with pink along edge of pale pink tint. white (7 ca). definite pink tint. grow Soluble pigment Soluble pigrner1t- Soluble pigment- Soluble pigment faint yellow. faint yellow. faint pinkish-yellow.

faint yellow.

Czapek-Dox agar Reverse-crearn. Reverse-white. Reverse cream. Moderate growth, fiat Aerial mycelium-flat, Aerial myceliurnflat, Aerial myeelium-flat, and colorless with powdery, ivory with powdery, ivory. powdery, mottled areas of yellow definite pink tint. No soluble pigment. pink, cream and vegetative growth. Soluble pigment white (7 ca). Aerial myceliu m faint yellow. Soluble pigment sparse, whitish.

faint yellow. N0 soluble pigment.

Glycerol- Reverse-yellow- Reverseorange. Reverseorange. Reverse-cream to asparaginaso brown. Aerial mycelium-fiat, Aerial myeeliurn-flat, ellow. agar. Aerial myceliumflat, grainy, pale beige powdery, pale rose- Aerial myceliumpowdery, very pale, with white edging. eige. moderate, pale cream. flesh pink. Soluble pigment- Soluble pigmel1t- Soluble pigment Soluble pigmenttan. tan. tan. light pinkish-tan.

Synthetic agar Smooth, spreading colorless growth. Aerial mycelium thick, cottony mass covering surface, scashell pink.

Yeast extract Reverse-orange. Reverse-mottled Reverse-orange. Reversetan.

dextrose agar. Aerial myceliumflat, yellow and brown. Aerial mycelium-flat, Aerial mycelium-light powdery, pinkish- Vegetative growth velvety, crearngrowth white in color. cream (approx. 3 ca). tan. colored in center of Spreading fan-shaped Spreading fan-shaped Aerial mycelium growth streak, pink areas of creamareas of creammoderate, white. on sides, and white colored vegetative colored vegetative Soluble pigrnenttan. on edges. growth with no growth with no Soluble pigment tal1. aerial mycelium aerial mycelium extending from main extending from main mass of growth. mass of growth. Soluble pigment-- Solublo pigment-tan. light tan.

Glucose agar Growth restricted,

glossy, bufi-colored, lichenoid margin.

TABLE 1-Continued Single-colony isolates derived from culture MA-2898 Original Culture Mad-2898 (ATCC 21096) MA-2911 (ATOO 21097) MA-2912 (ATCC 21098) MA-2913 (ATCC 21099) Strtptomjl/ces fradiae Skim milk Ring of moderate Ring of moderate Ring of moderate Ring of light vegetative vegetative growth, vegetative growth, vegetative growth, growth creamy. yellow. yellow. y llow. No aerial myeelium. Aerial myeelium- Aerial mycelium- Aerial mycelium- Partial peptonization.

sparse, creamy. sparse, creamy. sparse, creamy. Soluble pigment- Partial peptonization. Partial peptonization. Partial peptonization. pinkish cream. Soluble pigment Soluble pigment Soluble pigment pH 7.1.

cream to light tan. cream to light tan. cream to light tan. pH 8.0. pH 7.9. pH 7.8.

Micro-aerophilic Surface growth-good. Surface growthgood. Surface growth-good. Surface growthgood. Aerobic.

growth (yeast Depth of growthjn Depth of growth in Depth of growth in Depth of growth in extract-dextrose stab-5 mm. stab5 mm. stab 515 mm. stab515 mm. agar stab-45 mm. Aerobic. Aerobic. Aerobic. Aerobic.

depth).

Pigment solubility The growth is pigmented. In addition, a small amount of non-melanoid soluble pigment is released. The pigment formed is On some media, the soluble pigment is limited to the area immediately surrounding growth. Does not have indicator properties.

These antibiotic-producing strains were also tested for their ability to utilize or assimilate various carbohydriates. For this purpose, the microorganisms were grown in basal synthetic medium (Pridham and Gottlieb) containing 1% of the carbohydrate at 28 C. for three weeks. Table 2 shows the utilization of these carbohydrate sources by the Antibiotic 833A-producing strains: indicating good growth, poor growth, and no growth On the particular carbohydrate.

TABLE 2.CARBOEYDRATE UTILIZATION [Carbohydrate utilization (Pridharn & Gottlieb Basal Synthetic Medium i% carbohydrate)] Single-colony isolates derived The characteristics described in the foregoing tables were used in following the keys to the species of Streptomyces published in Bergeys Manual of Determinative not soluble.

Bacteriology, 7th Edition, pages 745-753, and The Actinomycetes, volume II, pages 156-164. Comparison of the detailed characteristics of the culture and its variants shows that the cultures resemble the dcscription of the type culture of Streptomyces fradz'ae in all significant properties. Accordingly, these Antibiotic 833A-producing cultures have been assigned the species designation Streptomyces fradiae.

Another species of Streptomyces fradiae producing the new antibiotic of this invention which was also isolated from soil is designated as MA-2915 in the culture collection of Merck & Co., Inc., Rahway, N]. This culture has also been placed on permanent deposit with the culture collection of the Northern Utilization Research and Development Division of the US. Department of Agriculture at Peoria, 111., and has been assigned the culture number 0 NRRL-3417.

The new antibiotic of this invention is also produced by growing, under controlled conditions, other previouslyunknown strains of Streptomyccs. Thus, other strains of Streptomyces also isolated from soil and identified in the Merck & Co., Inc., culture collection as cultures MA- 2867, 2903, 2916, 2917, 3270, 3272 and 3269 produce the new antibiotic of this invention.

The cultural characteristics of strains MA-2867, 2903, 2916, 3270, 2917 and 3272 were found to be very similar. The characteristics of four of these cultures, namely, MA2916, 2917, 2903, 2867 and 3269 are shown in Table 2.

TABLE 3 [Cultural characteristics of Streptomyces species which produce (cis-l,2-epoxypropyl)phosphonic acid] MA-2916 MA-2917 MA-2903 MA-2867 Mil-3269 Morphology Loops and spirals (open No sporulation found Hoolrs, loops and short Loops and spirals (both Sporophores branched,

and closed); spores on any medium used; sp rals; spores oval to open and closed); straight, produced oval to spherical0.9 very little visible cyl1nd rical 0.9 X 1.2- spores 0.9-1.2 x 1.2-1.7 in tufts; spores x 1.2-1.7 11.; in chains aerial mycelium. 1.7 p; 1n chains of p; in chains of more cylindrical-1.2 x 1.7 n;

of more than 15.

Sporulation seen on tomato paste-oatmeal, glycerol-asparagiue, and egg albumin agars.

Sporulation seen on in chains oi more than 10.

than 15.

Sporulation seen on tomato paste-oatmeal, tomato paste-oatmeal, glyeerol-asparagino, glyccrol-asparagine, and egg albumin and egg albumin ngars. agars.

TABLE 1 Continued Single-colony isolates derived from culture MA-2898 Original Culture MA-2898 (ATCC 21096) MA-29ll (ATCC 21097) MIL-2912 (ATCC 21098) MA-2913 (ATCC 21099) Streptomycesfradiae Glucose broth Dense, narrow, orangecolored ring, abundant, flaky, colorless sediment.

Starch agar Reverse-cream. Aerial mycelium-fiat, powdery, very pale flesh pink edged with cream. Soluble pigmentiaint y low. Starch is hydrolyzed.

Reverse-cream.

Aerial mycelium fiat,

powdery, pale beige edged with cream.

Soluble pigment-faint tan. Starch is hydrolyzed.

Reverse-cream.

Vegetative growth spreading, almost colorless.

Aerial mycelium moderate, pinkish- Soluble pigment-faint pink. Starch is hydrolyzed.

Spreading, colorless growth Starch is hydrolyzed.

Nutrient starch agar- Vegetative growthcream.

Reverse-yellow.

Aerial myceiium-pale flesh pink.

Starch is hydrolyzed.

Vegetative growth cream. Reverseyellow. Aerial mycelium cream with pink tint. Starch is hydrolyzed.

Vegetative growtl1 cream. Reverse-yellow. Aerial myceliumpink edged with cream. Starch is hydrolyzed.

Vegetative growthcream, spreading. Reversecoiorless to cream. Aerial mycelium moderate, creamy white. Starch is hydrolyzed.

Gelatin stab Cream-colored Cream to orange Creamcolored Cream-colored Cream-colored to vegetative growth. vegetative growth. vegetative growth. vegetative growth. brownish, dense No soluble pigment. No soluble pigment. No soluble pigment. No soluble pigment. growth on liquid I Gelatin liquefied. Gelatin liquefied. Gelatin liquefied. Gelatin liquefied. medium.

Nutrient gelatin Vegetative growth- Vegetative growth- Vegetative growth- Vegetative growthagar plates. cream. white to cream. white to cream. cream to yellow,

Aerial mycelium- Aerial mycelium- Aerial mycelium spreading.

moderate, creamy moderate, white. moderate, white. Aerial mycelium White. No soluble pigment. No soluble pigment. sparse, whitish. No soluble pigment. Gelatin liquefied. Gelatin liquefied. No soluble pigment. Gelatin liquefied. Gelatin liquefied.

Potato plugs Vegetative growth Vegetative gr0wth Vegetative growth Vegetative growth Restricted, orangeorange, wrinkled. orange, wrinkled. orange, wrinkled. burnt orange, dry, colored growth. Aerial mycelium Aerial mycelium Aerial myceliumdeep wrinkled.

moderate, deep moderate, deep cream, droplets of No aerial mycelium. cream, droplets of cream. orange exudate. Moderate darkening of orange exudate. Moderate darkening of Moderate darkening of plug. Moderate darkening of plug. plug.

plug.

Loefller blood Vegetative growthtan. Vegetative growth-tan. Vegetative growth-tan. Vegetative growthtan.

serum. No aerial mycelium. N o aerial mycelium. No aerial mycelium. No aerial mycelium.

Light tan pigment. Light tan pigment. Light tan pigment. Light tan pigment. Moderate liquefaction. Weak liquefaction. Weak liquefaction. Liquefaction very slight,

1 any.

Calcium malate Vegetative growth- Vegetative growth Vegetative groWth- Vegetative growthagar. colorless. colorless. cream. colorless.

Aerial mycelium- Aerial mycclium Aerial mycelium-- Aerial mycelium cream with pink tint. moderate, cream with very pale flesh pink. sparse, creamy. No soluble pigment. pink tint. N0 soluble pigment. No soluble pigment. Clear area surrounding No soluble pigment. Clear area surrounding Clear area surrounding growth. Clear area surrounding growth. growth.

growth.

Beptone-iron-yeast Vegetative growth Vegetative growth- Vegetative growth Vegetative gorwth extract agar. cream. cream. cream. cream.

No aerial mycelium. No aerial mycelium. No aerial mycelium. No aerial mycelium. No HzS production. No IIzS production. No HzS production. No H28 production. No soluble pigment. N0 soluble pigment. No soluble pigment. No soluble pigment.

Production oi ni- Negative Negative Negative Negative Nitrites not produced trites from nitrates. from nitrates.

Growth at 50 C.. No growth No growth No growth N 0 growth (1 week-yeast extract-dextrose agar) Tyrosine agar Vegetative growth- Vegetative growth Vegetative growth Vegetative growthtan with rose tint, moderate, tan with moderate, tan with poor, colorless, spreadmoderate. rose tint. rose tint. ing. Aerial mycelium- Aerial mycelium Aerial mycelium Aerial mycelium beige, moderate. beige moderate. light rose-beige. very sparse. Soluble pigment Soluble pigment- Soluble pigment- Soluble pigment light rosy-brown light rosy-brown. rosy-brown. very light rosybrown.

Skim milkagar- Vegetative growth Vegetative growth Vegetative growth Vegetative growthtan, smooth, moisttan, smooth, moisttan, smooth, moisttan, smmoth, moistappearing. appearing. appearing. appearing. No aerial mycelium. No aerial mycelium. No aerial mycelium. No aerial mycelium. Soluble pigment- Soluble pigment- Soluble pigment- Soluble pigmenttan. tan. tan. tan. Clear area showing Clear area showing Clear area showing Clear area showing hydrolysis of casein. hydrolysis of casein. hydrolysis of casein. hydrolysis of casein Litmus milk Ring of moderate Ring of moderate Ring of moderate Ring of light vegetative Faint, creamcolored vegetative growth, vegetative growth, vegetative growth, growth, creamring; coagulated, pepgrayish cream to tan. grayish cream to tan. grayish cream to tan. colored. tonized, becoming Aerial mycelium Aerial mycclium Aerial mycelium- No aerial mycelium. alkaline.

sparse, whitish. sparse, whitish. sparse, whitish. Partial peptonization. Partial peptonization. Partial peptonization. Partial peptonization. pH 7 .4. pH 8.2. pH 8.1. pH 8.1.

TABLE 3Continued Mil-3269 Tomato pasteoatmeal agar.

Vegetative growth reverse-orange.

Aerial mycelium mixture of white and blue-gray.

Soluble pigmentlight brown.

Vegetative growth brown. Aerial mycelium none. Soluble pigment-tan.

Vegetative growthbrownish orange.

Aerial mycelium cream edged with blue-gray.

Soluble pigmenttan.

Vegetative growth brownish orange. Aerial myceliummixture of white and blue-gray (approx. 1

Soluble pigment-tan.

Vegetative growth reverse-brown to tan.

Aerial mycelium grainy, light to dark gray. Soluble pigment-light tan.

Glycerol-asparagine a ar.

Vegetative growth reverse-brown.

Aerial myceliummoderate; mixture of white and blue-gray.

Soluble pigmenttan.

Vegetative growth Aerial mycelium none.

Soluble pigmenttan.

Vegetative growth reverse-reddish brown.

Aerial mycelium mixture of white and blue-gray [gray(d)].

Soluble pigment reddish brown.

Vegetative growthtan Aerial myceliummoderate, light gray.

Soluble pigment-light tan.

Czapek-Dox agar 7 (sucrose nitrate).

Vegetative growth reverse-yellow-tan.

Aerial mycelium scant, white along edges. Soluble pigment-tan.

Vegetative growthtan.

Aerial mycelium none.

Soluble pigment-tan.

Vegetative growth golden yellow.

Aerial mycelium moderate, cream.

Soluble pigmenttan.

Vegetative growth reverse-deep cream.

Aerial mycelium moderate, cream to white. Soluble pigment-tan.

Vegetative growth thin, colorless.

Aerial myceliumpale gray.

Soluble pigment none.

Egg albumin agar...

Vegetative growth reverse-reddish brown.

Aerial mycelium Vegetative growth tan Aerial mycelium none Vegetative growth cream.

Aerial myceliummixture of white and Vegetative growth reverse-pinkish tan.

Aerial mycelium mixture of white and Vegetative growth flat, spreading, reverse-grayish.

Aerial mycelium moderate, white to Soluble pigmenttan. blue-gray. Areas of bluegray. grainy, light to dark blue-gray. blue-gray along edge Soluble pigment gray. Soluble pigment [gray (e)]. pinkish tan. Soluble pigment reddish brown. Soluble pigment none. none.

Calcium malate Vegetative growth Vegetative growth- Vegetative growth Vegetative growth agar.

colorless to cream. Aerial mycelium cottony, bluish-gray and white.

Soluble pigment none.

Utilization of malatepartial clearing of area along growth.

tan. Aerial myceliumone.

Soluble pigment none.

Utilization of malate partial clearing of area around growth.

Vegetative growth tan Aerial mycelium brownish White.

Soluble pigmentnone.

Utilization of malate clear zone around growth.

reversecream.

Aerial myceliummoderate, blue-gray.

Soluble pigment none.

Utilization of malate clear zone around growth.

fiat, spreading, colorless.

Aerial mycelium grainy, medium gray.

Soluble pigment none.

Utilization of malateelear zone surroundin g growth.

Nutrient agar plates.

Vegetative growth moderate, tan.

Aerial mycelium sparse, whitish.

Soluble pigment-none.

Vegetative growth moderate, tan. Aerial mycelium one. Soluble pigmentnone.

Vegetative growth spreading, tan.

Aerial mycelium grayish white.

Soluble pigment-none.

Vegetative growthtan.

Aerial mycelium grainy, light to medium gray edged with white.

Soluble pigment-very light tan.

Nutrient tyrosine Vegetative growth Vegetative growth- Vegetative growth- Vegetative growth Vegetative growth agar. tan to grayish tan. brown. tan to grayish tan. tan. tan.

Aerial mycelium Aerial mycelium Aerial mycelium Aerial mycelium Aerial mycelium sparse, brownish. none. grayish cream. gray. thin, gray edged with Soluble pigment Soluble pigment Soluble pigment Soluble pigment white.

grayish tan edging grayish tan edging grayish brown. brownish gray edging Soluble pigment-light growth. growth. Clear zone around growth. tan.

Clear zone around Clear zone around growth. Clear zone around Clear zone around growth. growth. growth. growth.

Yeast extract Vegetative growth Vegetative growth (Not tested on this Vegetative growth Vegetative growth dextrose agar. brown. brown. medium.) reserve-dark brown. light brown.

Aerial mycelium Aerial myceliumnone. Aerial mycelium Aerial mycelium scant, light gray and Soluble pigment light gray to white. sparse, grayish white. white. brown. Soluble pigment Soluble pigment-light Soluble pigment-none. brown. brown.

Starch agar Vegetative growth Vegetative growth Vegetative growth Vegetative growth- Vegetative growth (synthetic). reverse-cream to ligyt tan, transparent. colorless. reverse-tan. light grayish tan to colorless. Aerial mycelium Aerial mycelium Aerial myceliumwhite.

Aerial mycelium none. grayish blue. cottony, grayish blue Aerial mycelium grayish blue to white. Soluble pigment Soluble pigment- (l9dc). grainy, light to Soluble pigment slight browning of slight darkening of Soluble pigmenttan. medium gray.

slight yellowing of medium. medium. (edging growth). Soluble pigmentnone. medium.

Nutrient gelatin Vegetative growth Vegetative growth Vegetative growth Vegetative growth Vegetative growth plates. tan, moderate. an. tan. tan. tan,

Aerial mycelium Aerial mycelium- Aerial mycelium- Aerial mycelium Aerial mycelium n ne none. sparse, whitish. sparse, grayish white. none.

Soluble pigment-tan. Soluble pigment- Soluble pigment Soluble pigment tan. Soluble pigmentvery Liquefaction-complete. brown. brown. Liquefactionco1nplete. light tan.

Liquefaetion Liquefaction-- Liquefactioncomplete. complete. good,

Gelatin stabs Vegetative growth Vegetative growth Vegetative growth Vegetative growth Vegetative growthcream, suspended cream, suspended tan flales at bottom of cream, suspended cream to'tan ring, throughout medium. throughout medium. tube... throughout medium. flakes on bottom of Soluble pigment- Soluble pigment Soluble pigment Soluble pigment of tube.

greenish brown. greenish'brown. brown. greenish brown. Aerial mycelium Liquefaetion- Liquefaction- Liquefaction-1 Liquefaction-w non M V l complete. complete. complete. complete. Soluble pigment-very a light tan.

Liquefactioncomplete.

Nutrient starch Vegetative growth Vegetative growth Vegetative growth Vegetative growth I Vegetative growthagar. tan. tan. cream; reverse-cream tan. tan. Y,

Aerial mycelium Aerial mycelium to yellow. Aerial mycelium Aerial mycelium" grayish white. Aerial mycelium grayish white. gray edged with white Soluble pigment-light tan. Hydrolysismoderate.

none.

Soluble pigmentlight brown.

Hydrolysismoderate.

moderate, grayish white. Soluble pigmentlight brown. Hydrolysismoderate.

Soluble pigment-tan edging growth. Hydrolysismoderate.

Soluble pigment-very light tan.

Hydrolysis-.moderate.

TABLE 3Continued Mix-2916 MIA-2917 MIX-2903 MA-2867 MA-3269 Skim milk agar Vegetative growth- Vegetative growth- Vegetative grwth Vegetative growth Vegetative growth plates. grayish brown. brown. center brown, edges reverse-reddish tan.

Aerial myceliu1n Aerial myceliurngray. brown. Aerial myceliui'n none. none. Aerial myceliuin Aerial rnyeelium grayish white to light Soluble pigment Soluble pigmcnt none. brown and gray. gray.

reddish brown. brown. Soluble piginent Soluble pigmentdark Soluble pigment-light Casein hydrolysis Casein hydrolysisdark reddish brown. reddish brown tan.

good. good. Casein liydrolysis edging growth. Casein hydrolysisgood. Casein hydrolysis good.

moderate.

Skim milk Vegetative growth- Vegetative growtli- Vegetative growth Vegetative growth Vegetative growth moderate, brown moderate, tan to heavy, brown ring. moderate to heavy thin growth ring, ring. brown ring. Aerial inyeclium brown ring. cream to tan.

Aerial mycelium- Aerial myceliumbrownish gray. Aerial mycelium- Aerial myceliumsparse, whitish. none. Soluble pigment sparse, whitish. none.

Soluble pigment- Soluble pigment dark brown. Soluble pigment- Soluble pigment-tan.

cream to brown cream to brown Peptonization. dark brown. Soft coagulum followed (layered). (layered). pH 6.1. Partial peptonization. by peptonization.

Partial peptonization. Partial peptonization. pH 6.4. pH 7.7.

soft coagulum. pH 6.3.

Litmus milk Vegetative growth Vegetative growth Vegetative growth Vegetative growth- Vegetative growthheavy, brown ring. moderate, brown dark brown moderate dark brown moderate thin growth ring.

Aerial myceliumpartial ring. to heavy ii'ng. ring. Aerial myceliumnone.

sparse, whitish. Aerial mycelium-none. Aerial myeelium- Soluble pigment- Solft coagulum Soluble pigment Soluble pigment grayish white. brown. followed by reddish brown. reddish brown. Soluble pigment Peptonization. peptonization.

Peptonization. Peptonization. dark brown. pH 6.8. pH 8.2.

pH 6.8. pH 6.9. Peptonization.

Carbohydrate utilization (Pridham & Gottlieb Basal Synthetic Medium-1% carbohydrate) No sugar No growth N 0 growth N0 growth N 0 growth Good growth.

Very limited growth or none. Do. P Moderate growth. Inositol No growth i (Pp) +(P) Very limited growth or none. Good growth.

Do. Do. Very limited growth or none. Moderate growth. Good growth. Very limited growth or none. Rhamncse (P) (PD) (P) Good growth.

Potato plug Vegetative growth Vegetative growth- Vegetative growth- Vegetative groWth- Vegetative growthgray. dark tan. darglflanl, heavily grayish tan to gray. wrinkled, tan.

wri e Aerial mycelium- Aerial mycelium Aerial mycelium Aerial myceliuni Aerial mycelium none. none. light gray to bluenone. none. gray. Soluble pigment Soluble pigment Soluble pigment Soluble pigment Soluble pigmentgray-brown. gray-brown. brown. gray. srime browning of p ug.

Peptone iron-yeast Vegetatiine growth- Vegetative growth- Vegetative growth Vegetative growth Vegetative growth extract agar. dark gray. dark gray. dark gray to black. dark gray. tan.

Aerial myoelium Aerial myceliuln Aerial mycelium Aerial mycelium Aerial Inycelium none. none. none. none. none.

Soluble pigment Soluble pigment- Soluble pigment- Soluble pigment- Soluble pigmentblack. black. almost black. black. none.

Loefiiers blood Vegetative growth- Vegetative growth Vegetative growth- Vegetative growthserum slants. gray. gray. dark gray. gray.

Aerial mycelium Aerial mycelium Aerial mycehum Aerial mycelium none. none. none. none.

Soluble pigment Soluble pigment- Soluble pigment- Soluble pigmentdark brown. dark brown. gray to black. grayish-brown.

Liquefaction good. Liquefaction good. Liquefaction none. Liquefaction-mono.

Reduction of No reduction to N 0 reduction to No reduction to Noreduction to Positive.

nitrates (organic nitrites. nitrites. nitrites. nitrites. medium).

Microaerophilic Good surface growth. Good surface growth. Good surface growth Good surface growth Good surface growth growth (yeast Isolated colonies Isolated colonies and along upper and along upper and along of stab extract-dextrose along upper 10 mm. along stab line for 5 mm. of stab hne. 5 mm. of stab line. line. agar stab of stab line. 15 mm.

mm. depth).

Temperature Good growth at 28 C. Good growth at 28 0. Good growth at 28 0. Good growth at 28 0. Good growth at 28 C.

No growth at C.

No growth at 50 C.

N 0 growth at 50 C.

N 0 growth at 50 C. and 37 C No growth at C.

ration of America.

(1') :Soluble piginent-liglit tau. (ly):S0li1blc pigment-yellowish tan.

(Pp) Soluble pigincntpinkisli tan. On triplicate plates had i,

Based upon these cultural characteristics, it has been conlished type description of Streptomyces viridochromogenes cluded that these cultures should be classified as members in lacking gray to black mycelial coloration when grown of the species Streptomyces viridochromogenes, although on synthetic agar. Since this is the only significant difall of the above-mentioned strains differ from the pubference, it is not considered to be adequate to justify assigning a new species name to these cultures. The viridochromogenes section of the Streptomycetes includes a group of closely-related cultures. Some authorities apply separate species names, inter alia S. chartreusz's, S. cyaneus, as well as 'bicolor, coeruleofuscus, coerulcrorubidis, coerulescens and longisporus, which Russian taxonomists place in the Actinomyces, equivalent to the Streptomyces group of other scientists. Cultures MA- 2903, 2867, 2916, 2917 and 3270 have been placed on permanent deposit with the culture collection of the :Northern Utilization Research and Development Division of the US. Department of Agriculture at Peoria, Ill., where they are available as cultures NRRL-3413, 3414, 3415, 3416 and 3427, respectively. Oilture MA-2916 has also been deposited in the culture collection of the American Type Culture Collection, which it is available as ATCC-21240.

The characteristics of culture MA-3269 are also shown in Table 2. The culture clearly is a member of the series Cinereus as defined in The Actinomycetes, vol. II, S. A. Waksman. Based on the sporophore morphology, the culture falls within the section Rectus-Flexibilis, as described by Pridham, Hesseltine, and Benedict, in Applied Microbiology 6, 52 (1958). It differs in some aspects from all previously described species of Streptomyces. It is most closely related to Streptomyces wedmorensis. A relatively rapid peptonization of milk and the production of a soft coagulum are significant differences but are not considered sufficient to establish a new species. For this reason, the culture has been assigned to the species S. wedmorensis. This culture has been deposited in the American Type Culture Collection, where it is available as ATCC- 21239 and in the A.R.S. Collection of the Norther Utilization and Research and Development Division of the U.S. Department of Agriculture at Peoria, 111., where it is available as NR'RL-3426.

The above descriptions of the microorganisms producing Antibiotic 833A are given as illustrative of suitable strains of microorganisms, which can be used in the production of the antibiotic, but it is to be understood that the present invention is not to be limited to organisms answering these particular descriptions. The present invention also contemplates the use of other microorganisms including strains of Streptomyces either isolated from nature or obtained by mutation of these organisms such as those obtained by natural selection or those produced by mutating agents, for example, X-ray irradiation, ultraviolet irradiation, nitrogen mustards, and the like.

' The new antibiotic of this invention is produced during the aerobic fermentation of suitable aqueous nutrient mediums, under controlled conditions described hereinafter by (cis-1,2-epoxypropyl)-phosphonic acid producing strains of microorganisms. Aqueous mediums, such as those emplyoed for the production of other antibiotics, are suitable for producing this new antibiotic. Such mediums contain sources of carbon and nitrogen assimilable by the microorganism and inorganic salts. In addition, the fermentation mediums contain traces of metals necessary for the growth of the microorganism which are commonly supplied as impurities incidental to the addition of other constituents of the medium.

In general, canbohydrates such as sugars, for example, sucrose, maltose, fructose, lactose, and the like, and starches such as grains, for example, oats and rye, corn starch, cornmeal, and the like, can be used either alone or in combination as sources of assimilable carbon in the nutrient medium. The exact quantity of the carbohydrate source or sources utilized in the medium will depend in part upon the other ingredients of the medium, but it is usually found that an amount of carbohydrate between about 1 and 6% by weight of the medium is satisfactory. These carbon sources can be used individually or several such carbon sources may be combined in the medium.

Satisfactory nitrogen sources include myriad proteinaceous materials such as various forms of hydrolysates of casein, soybean meal, corn steep liquor, distilled solubles, yeast hydrolysates, tomato paste, and the like. The various sources of nitrogen, either alone or in combination, are used in amounts ranging from about 0.2-6% by weight of the aqueous medium. Examples of mediums suitable for the production of (cis-l,2-epoxypropyl)-phosphonic acid are shown in the examples which follow.

The fermentation using the (cis-1,2-epoxypropyl)- phosphonic acid producing microorganisms can be carried out at temperatures ranging from about 25-38 C. For optimum results, it is most convenient to conduct these fermentations at temperatures between 26-30 C. The pH of the nutrient mediums suitable for growing the Streptomyces and producing the antibiotic can vary from about 5.5-7.5.

Although the new antibiotic of this invention is produced by both surface and submerged cultures, it is presently preferred to carry out the fermentation in the submerged state. Small scale fermentations are conveniently carried out by placing suitable quantities of nutrient medium in flasks, sterilizing the flasks and contents by heating to C., inoculating the flasks with either spores or a vegetative cellular growth of a (cis-1,2- epoxypropyl) phosphonic acid producing strain of Streptomyces, loosely stoppering the necks of the flasks with cotton, and permitting the fermentation to proceed in a constant temperature room at about 28 C. on a shaker for 3-5 days. For larger scale work, it is preferable to conduct the fermentation in suitable tanks provided with an agitator and a means of aerating the fermentation medium. In this method, the nutrient medium is made up in the tank and sterilized by heating at 120 C. After cooling, the sterilized medium is inoculated with a suitable source of vegetative cellular growth of the Streptomyces and the fermentation is permitted to proceed for 2-4 days while agitating and/or aerating the nutrient medium and maintaining the temperature at about 28 C. This method of producing (cis-1,2-epoxypropyl)-phosphonic acid is particularly suited for the preparation of large quantities of the new antibiotic.

In carrying out the production of the antibiotic in the submerged state, a small amount of a suitable anti-foam agent such as soybean oil, castor oil, 1% octadecanol in mineral oil, or a polymerized propylene glycol such a Polyglycol 2,000 can be added to the fermentation broth to control excessive foaming during the fermentation.

Assay (Cis-1,2-epoxypropyl)phosphonic acid is conveniently assayed by a disc-plate procedure using Proteus vulgaris MB-838 (ATCC 21100 and NRRL B-336l) as the test organism. The test culture is maintained as a slant culture on nutrient agar (Difco) plus 0.2% yeast extract (Difco). The inoculated slants are incubated at 37 C. for 18-24 hours and stored at refrigerator temperatures for one week, fresh slants being prepared each week.

The inoculum for the assay plates is prepared each day by inoculating a 250 ml. Erlenmeyer flask containing 50 ml. of nutrient broth (Difco) plus 0.2% yeast extract (Difco) with a scraping from the slant. The flask is incubated at 37 C. on a shaking machine for 18-24 hours. The broth culture is then adjusted to 40% transmittance at a wavelength of 660 m using a Bausch & Lomb Spectronic 20 by the addition of 0.2% yeast extract solution to the growth. Uninoculated broth is used as a blank for this determination. 30 ml. of the adjusted broth is used to inoculate 1 liter of medium.

Nutrient agar (Difco) plus 0.2% yeast extract (Difco) is used as the assay medium. This medium is prepared, sterilized by autoclaving, and allowed to cool to 50 C. After the medium is inoculated, 10 ml. is added to sterile petri dishes and the medium is allowed to solidify.

The activity is expressed in terms of units, a unit being defined as the concentration of the antibiotic per ml. which on a /2 inch paper disc will produce a zone diameter of 28 mms. Four concentrations of 833A are employed for the preparation of the standard curve, namely, 0.3, 0.4, 0.6 and 0.8 units per ml.; each concentration being obtained by the dilution in tris (hydroxymethyl)aminomethane buffer adjusted to pH 8.0. Four discs are placed on each of the five plates for the preparation of the standard curve, each plate containing one disc of each of the four concentrations of antibiotic shown above. The plates are incubated for 18 hours at 37 C. and the diameters of the zones of inhibition in mms. are measured. An average zone diameter for each concentration is calculated, from which a standard curve is prepared on semi-log graph paper. The slope of the line obtained is between 4 and 5.

Samples of the antibiotic to be assayed are diluted in 0.05 M buffer at pH 8.0 to an appropriate concentration. Discs are dipped into the test solution and placed on the surface of the assay plate; two discs for each sample are normally placed on one plate opposite to one another. Two discs dipped into 0.4 units per ml. (cis-1,2-epoxypropyl)-phosphonic acid solution are placed on the plate in an alternate position to the sample. The plates are incubated at 37 C. for 18 hours and the zone diameters in mms. are determined. The potency of the sample is determined by means of a nomograph or from the standard curve. 1 mg. of pure (cis- 1,2-epoxypropyl)-phosphonic acid contains 357 units.

Properties of (cis-l,2-epoxypropyl)posphonic acid HaC This substance is an acidic compound which is now believed to be more properly named as (cis-1,2- epoxypropyl)phosphonic acid in accordance with present chemical nomenclature practice; the indicating, as does the letter I, that this phosphonic acid rotates planepolarized light in a counterclockwise direction (to the left as viewed by the observer) when the rotation of its disodium salt is measured in water (5% concentration) at 405 111111.. The designation cis used in describing the 1,2-epoxypropylphosphonic acid compound means that the hydrogen atoms attached to carbon atoms 1 and 2 of the propylphosphonic acid are on the same side of the oxide ring.

The structural formula of this antibiotic substance has been shown in the planar formula for the sake of convenience. However, the antibiotic can also be depicted spatially as follows:

The free acid in crystalline form is a white solid melting at or above about 94 C. when placed in a bath at this or higher temperatures.

The (cis-1,2-epoxypropyl)-phosphonic acid is a water soluble acidic substance which reacts with salt forming substances, such as inorganic and organic bases, to form salts. Thus, upon reaction with alkali metal and alkaline earth metal hydroxides, carbonates, bicarbonates, the corresponding alkali metal or alkaline earth metal salt is obtained. Other metal salts such as silver, iron and the like can be similarly prepared by metathesis or in accordance with other methods well known to those skilled in this art. Similarly, salts of organic bases, such as primary, secondary and tertiary amines, for example, monoalkylamines, dialkylamines, trialkylamines,

alkyldiamines, and nitrogen-containing heterocyclic amines, are prepared in accordance with methods known in this art. The salts can be mono salts such as the monosodium salt obtained, for example, by reacting one equivalent of sodium hydroxide with one equivalent of acid; disalts obtained, for example, by reacting two equiv alents of sodium hydroxide with one equivalent of the acid; mixed disalts obtained by reacting one equivalent of the monosalt with one equivalent of a second base; diphosphonate salts obtained, for example, by reacting one equivalent of calcium hydroxide with two equivalents of the acid; mixed salts such as calcium hydrogen lactate obtained by reacting one equivalent of lactic acid with the calcium diphosphonate salt and the like.

Representative examples of salts of organic bases that might be mentioned are salts with amines such as ot-phenethylamine, diethylamine, quinine, brucine, lysine, protamine, arginine, procaine, ethanolamine, morphine, benzylamine, ethylenediamine, N,N'-dibenzylethylenediamine, diethanolamine, piperazine, dimethylaminoethanol, 2- amino-Z-methyl-l-propanol, thiophylline, esters of amino acids, N-methylglucamine and the like. If desired, the basic moiety of the salt can be biologically active amines such as erythromycin, oleandomycin, novobiocin and the like.

The salts of (cis-1,2-epoxypropy1)phosphonic acid which are pharmaceutically acceptable and substantially non-toxic are particularly valuable and can be utilized in preparing suitable dosage-unit pharmaceutical forms. All salts are useful as intermediates in preparing the free acid and for making other salts. The salts of opticallyactive bases, such as amines, are useful in the separation of optically-active enantiomers of the phosphonic acids.

(Cis-l,2-epoxypropyl)-phosphonic acid is very stable in aqueous solutions to alkaline conditions up to about pH 11 with ammonia or organic amines. Heating aqueous solutions at 100 C. for 15 minutes at a pH of 89 does not appear to inactivate the antibiotic. It is unstable below about pH 3. At pH 1.5, the half life at room temperature is less than 24 hours. Instability, even at a higher pH, has been observed in contact with sulfonic or phosphonic ion exchange resins. Incomplete recovery is obtained even with relatively short contact at pH 3 with resins of this type.

The compound is remarkably polar. Based on paper strip studies, the substance is similar to gluconic acid. It is not readily extracted, even in the presence of organic amines, by polar solvents such as phenol. Chromatography on cation exchange resins on the hydrogen cycle also indicates polar properties. Thus, on such resins, the antibiotic is much less retarded than citric acid. The polar nature of the new antibiotic is also indicated by the almost complete lack of adsorption from aqueous solutions by activated carbon at a pH of 5.

The antibiotic is not adsorbed on acidic clays, although alumina, both basic and acid washed, are good adsorbents for the antibiotic. From aqueous solution, the compound is strongly adsorbed at pH 5, but eluted at pH 9. At pH 9, adsorption is strong from 75% methanol-water, but relatively weak in 25% methanol-water. The addition of barium or calcium salts to aqueous solutions does not precipitate the antibiotic. The ammonium salt of the antibiotic appears to have limited solubility in anhydrous methanol, but the sodium salt is soluble in methanol-water.

The antibiotic containing fermentation broths produced in accordance with the procedures described herein have activities ranging from about 1-20 units per ml. when assayed in accordance with the standard assay using Proteus vulgaris described in the specification. The antibiotic can be purified and recovered in a purer form by a number of procedures. One such procedure comprises adsorbing the antibiotic on anion exchange resins, for example, resins composed of quaternary ammonium exchange or polyalkylamine groups attached to a styrenedivinyl-benzene polymer lattice. The adsorbed antibiotic is readily eluted from the resin adsorbate with aqueous or aqueous-alcoholic solutions of salts such as ammonium chloride, sodium chloride, sodium acetate and the like. The eluate so obtained can be further purified, if desired, by other purification procedures. Thus, the eluate can be purified by passing it through a bed of polyacrylamide gel having pore sizes allowing the fractionation of sub stances having molecular weights between 200 and 2,000. Purification of the antibiotic can also be achieved by passing the impure antibiotic solution through a strongly acidic cation exchange resin composed of nuclear sulfonic acid exchange groups attached to a styrene-divinylbenzene polymer lattice, and developing the resin with water. These purification procedures are described in detail in the pending application of Thomas W. Miller, Ser. No. 699,376, filed Jan. 22, 1968.

Alternatively, the antibiotic can be purified by adsorption on alumina; either basic or acid washed alumina being suitable for this purification. The adsorbed antibiotic can be eluted from the alumina most conveniently by aqueous or aqueous-alcoholic ammonium hydroxide solutions having a pH of about 11.2 and fractionally collecting the eluate. Purification of impure solid fractions containing the ammonium salt of (cis-l,2- epoxypropyl)phosphonic acid can also be affected by dissolving such material in methanol, adding an equal volume of n-butanol, evaporating off the methanol, filtering off any butanol insoluble material, and recovering a butanol solution containing the ammonium salt of the antibiotic of enhanced purity. The ammonium salt can then be obtained in solid form by evaporating the butanol solution to dryness under reduced pressure. Alternatively, the ammonium salt can be extracted from the butanol solution with water to obtain an aqueous solution of the ammonium salt of (cis-1,2-epoxypropyl)-phosphonic acid. The calcium salt of the antibiotic is produced by adding calcium hydroxide to the aqueous solution of the ammonium salt and warming the resulting solution under reduced pressure. Alternatively, the calcium salt is also obtained by passing a solution of another salt of the antibiotic over a cation exchange resin on the calcium cycle. The calcium salt crystallizes from aqueous solutions having a concentration of mg./ml. upon standing or with agitation. These purification procedures are described in more detail in the pending application of Louis Chaiet, Ser. No. 699,377, filed Jan. 22, 1968.

(Cis-1,2-epoxypropyl)-phosphonic acid and its salts are useful antimicrobial agents, which are active in inhibiting the growth of both gram-positive and gramnegative pathogenic bacteria. This antibiotic and particularly its salts, are active against Bacillus, Escherichia, Staphylococci, Salmonella and Proteus pathogens, and antibiotic-resistant strains thereof. Illustrative of such pathogens are Bacillus subtilis, Escherichia coli, Salmonella schottmuelleri, Salmonella gallinarum, Salmonella pullorum, Proteus vulgaris, Proteus mirabilz's, Proteus morganii, Stahphylococcus aureus and Stahpylococcus pyogenes. Thus, (cis-l,2-epoxypropyl)phosphonic acid and salts thereof can be used as antiseptic agents to remove susceptible organisms from pharmaceutical, dental and medical equipment and other areas subject to infection by such organisms. Similarly, they can be used to separate certain microorganisms from mixtures of microorganisms. Salts of (cis-l,2-epoxypropyl)phosphonic acid are also useful in the treatment of diseases caused by bacterial infections in man and animals and are particularly valuable in this respect, since they are active against resistant strains of pathogens. These salts are especially valuable, since they are effective when given orally, although they can also be administered parenterally.

Since the antibiotic and its salts are very active in inhibiting the growth of various species of Salmonella, it can be used as a disinfectant in washing eggs and areas subject to infection by Salmonella. The salts of (cisl,Z-epoxypropyl)phosphonic acid are also useful as bactericides in various industrial applications, for example, in inhibiting undesirable bacterial growth in the White water in paper mills and in paints such as polyvinyl acetate latex paint.

When (cis-1,2-epoxypropyl)phosphonic acid or its salts are used for combatting bacteria in man or lower animals, they may be administered orally in a dosage unit form such as capsules or tablets, or in a liquid solution or suspension. Alternatively, the antibiotic can be administered parenterally by injection. These formulations can be prepared using suitable diluents, extenders, granulating agents, preservatives, binders, flavoring agents, and coating agents known to those skilled in this art. Representative formulations can be prepared *by the following procedures:

In each of the above examples the active compound and the diluent are mixed to produce a uniform blend, which is then filled into No. 0 hard gelatin capsules, by hand or on a suitable machine, as required. The mixing and filling is preferably done in an area having a relative humidity less than 40%.

Tablets: Per tablet, mg. (1) Disodium (cis-1,2-epoxypropyl)phosphonate (anhydrous) (Equivalent to free acid 250mg.) 330 Dicalcium phosphate 192 Lactose, U.S.P 190 Cornstarch Magnesium stearate 8 (2) Calcium (cis-l,2-epoxypropyl)phosphon-ate (Equivalent to free acid 250 mg.) 352.5

Dicalcium phosphate 180.0 Lactose, U.S.P. 179.5 Cornstarch 80.0 Magnesium stearate 8.0

In the above examples, the active component is blended with the dicalcium phosphate, lactose and about half of the cornstarch. The mixture is granulated with a 15% cornstarch paste and rough-screened. It is dried at 45 C. and screened again through No. 16 screens. The balance of the cornstarch and the magnesium stearate is added and the mixture is compressed into tablets, approximately /2" in diameter, each weighing 800 mg.

Alternatively, the active component is blended with the dicalcium phosphate, lactose and one-half the cornstarch. The mixture is slugged on a heavy duty press to produce compacted tablet-like masses. These are broken down to a No. 16 mesh granule. The balance of the corn starch and the magnesium stearate are added and the mixture is compressed into tablets approximately /2" in diameter, each weighing 800 mg.

Lyo Form (For Injection) Per vial, mg. (1) Disodium (cis-1,2-epoxypropyl) phosphonate (anhydrous) (Equivalent to free acid 250 mg.) 330 Water-for-injection U.S.P. to make 5 ml.

19 (2) Disodium (cis-1,2-epoxypropyl)phosphonate (anhydrous) 330 Mannitol 500 Water-for-injection U.S.P. to make ml.

In the above examples, the active component, or the active component and the mannitol, are dissolved in sufficient water-for-injection in the ratio shown. The solution is filtered through Selas candles or Millipore membrane filters to sterilize. The solution is subdivided into sterile vials. The vials and contents are frozen, and the water is aseptically removed by lyophilization. The vials containing the sterile dry solid are aseptically sealed.

To restore for parenteral administration, 5 ml. of sterile water-for-injection is added to the contents of a vial.

. Per 1,000

Oral liquid forms Per 5 m1.

Disodium (cis-1 "-epox ropyD-phosphonato (anhydrous) 660 mg. 132.0 gm Sucrose 600.0 gm Glucose 250.0 gm Citric acid 13.0 gm. Sodium benzoate 1.0 gm. Concentrated orange oil 0.2 ml. Purified water U.S.P. to make 1,000.0 In]:

1 Equivalent to free acid 500 rug/5 ml.

Bottle 1: Per 100 ml. size Disodium (cis 1,2 epoxypropyl)-phosphonate (anhydrous) gm 6.60 Bottle 2:

Glycyrrhize Syrup U.S.P. ml 100 At time of use the contents of bottle 2 are transferred to bottle 1 and the contents dissolved. Each 5 ml. of the solution contains 330 mg. of disodium (cis-1,2- epoxypropyl)-phosphonate (anhydrous), equivalent to 250 mg. of the free acid.

The (cis-1,2-epoxypropyl)-phosphonic acid is preferably employed as an antibacterial agent in the form of a salt. Good results are obtained in the treatment and control of bacterial infections in adult humans by administration of from about A to 4 gm./day of (cis-1,2- epoxypropyl)-phosphonic acid equivalent, the actual weight being dependent on the particular salt. When the higher dose levels are used, the salt is chosen so that the cation is not unduly toxic at such levels. It will, of course, be understood that the optimum dose in any given instance will depend upon the type and severity of infection to be treated, and that smaller doses will be employed for pediatric use, all of such adjustments being within the skill of the practitioner in the field.

The (cis-1,2-epoxypropyl)-phosphonic acid compounds of this invention may be administered alone or in combination with other biologically active ingredients and especially with other antibacterial agents such as erythromycin, lincomycin, a penicillin, streptomycin, novobiocin, tylosin, gentamycin, neomycin, colistin, kanamycin, oleandomycin, triacetyl oleandomycin and spiromycin.

The following examples illustrate methods for producing and recovering the antibiotic of this invention.

20 EXAMPLE 1 To an agar slant culture of Streptomyces fradiae ATCC 21096 was added 10 ml. of sterile aqueous 0.9% sodium chloride solution. A cell suspension was prepared by scraping the surface of the growth, and 2 ml. of this suspension was added to a 250 ml. baflled Erlenmeyer flask containing 50 ml. of sterile FA nutrient medium of the following composition:

g./ liter Yeast extract l0 Glucose 10 MgSO .7H O 0.05 Phosphate buffer, 2 ml.

Water, balance.

91 g. of KH2POi +950 g. of NazHPOr made up to 1 liter in distilled Water.

The inoculated flask was incubated at 28 C. for 4 days on a 220 rpm. rotary shaker with a 2" throw.

To each of four 250 ml. bafiled Erlenmeyer flasks containing 50 m1. of sterile FD medium, prepared by diluting 20 g. of dry oatmeal and 20 g. of tomato paste to one liter with hot distilled water, was added 1.5 ml. of the vegetative culture prepared as described above. The inoculated flasks were incubated at 28 C. on a rotary shaker operating at 220 rpm. with a 2" throw and one flask removed after one day, one after two days, one after three days, and the remaining flask was removed after four days incubation. The entire contents of each flask was centrifuged at 5,000 r.p.m. for seven minutes and the broth was decanted from the solids. The supernatant broths were assayed and activity Was found in the broth harvested after 4 days of incubation. This broth gave an 11 mm. zone of inhibition in a Proteus vulgaris diffusion plate assay.

The presence of (cis-l,Z-epoxypropyl)-phosphonic acid in the broth was determined by agar diffusion assays performed with 7 mm. filter paper discs soaked in the broth and set on the surface of assay plates containing 5 ml. of nutrient agar (Difco) plus 0.2% yeast extract (Difco) medium seeded with the bacterial inoculum. The zones of inhibition were measured in mm. after overnight incubation at 25 C. The assays with the broth obtained after incubation for 4 days showed a zone diameter of 11 mm. on the plate seeded with Proteus vulgaris MB- 838 and 25 mm. on the plate seeded with Erwimz atroseptica MB-l159.

Paper strip chromatographic mobility was determined by developing strips of Whatman No. 3 filter paper spotted at the origin with the broth obtained after 4 days of incubation and developed with solvent K [70% isopropanol and 30% pH 6.0 phosphate buifer (0.01 M), or solvent C (2% sodium chloride in 75% aqueous methanol) Results were visualized by bioautography on thin nutrient agar plus 0.2% yeast extract medium plates seeded with bacterial inoculum. The mobilities which were recorded as the R.F. of the center of the zone of inhibition observed after overnight incubation were as follows:

R.F. in solvent system Bioautographic organism K C Proteus vulgaris MB-383 .36 73 Erwina atroseptica MB1159 28 .71

21 11.5 cm. under these conditions on a bioautographic plate s eeded with Erwirz aatroseptica.

EXAMPLE 2 To 10 ml. of sterile AO medium of the following composition:

Distilled water q.s., balance.

and having a pH of 7.2 before sterilization was added an agar'slant culture of Streptomyces fradiae MA2898 (ATCC 21096) on FA agar medium of the following composition: 7

Distilled water q.s., balance.

91 gdKHzPOi and 95 g. NazHPOr made up to 1 liter with distilled water.

, A 250 ml. baflied Erlenmeyer flask containing 50 ml. of sterile A medium was inoculated with 3 ml. of this cell suspension. The flasks were then shaken on a 220 r.p.m. rotary shaker with a 2" throw at 28 C. for 72 hours. The resulting inoculum was then used to seed each of a series of 2-liter baflled Erlenmeyer flasks containing 350 ml. of sterile FD medium, 10.5 ml. of the vegetative growth being used for each flask. The inoculated flasks were then incubated ona 145 r.p.m. rotary shaker having a 2" throw for 96 hours. The contents of flasks were then combined.

I The fermentation broth so obtained and having a pH of 6.6 was filtered with the aid of diatomaceous earth. The filtered broth was found to give a 26 mm. zone of activity when assayed with Proteus vulgaris as described above.

The PD medium was prepared by diluting 20.0 g. of dry. oatmeal and 20.0 g. of tomato paste to 1 liter with hot distilled water.

' EXAMPLE 3 A v lyophilized culture of Sterptomyces MA2898 (ATCC 21096) was used to inoculate 50 ml. of sterile FA Phosphate buifer, 2 ml. Distilled water q.s., balance.

1 91 g. KH2PQ4, and 05.0 g. Na2HPO4 made up to 1 liter with distilled water.

..The inoculated flask was then placed on a 220 r.p.m. rotary shaker with 2" throw and incubated for 48 hours at 28 C. and the resulting fermentation broth used to inoculate a second stage seed tank containing 50 ml. of the same medium in a 250 ml. of baffled Erlenmeyer flask using a 2% inoculum. The second stage seed flasks were also shaken on the rotary shaker at 28 C. for 2 days.

An inoculum of 10.5 ml. of this vegetative growth was then used to inoculate a 2-liter baflied Erlenmeyer flask containing 350 ml. of sterilized medium consisting of the following ingredients:

g./liter Dry oatmeal 20 Tomato paste 20 Distilled water q.s., balance.

22 These 2-liter flasks were incubated on a 145 r.p.m. rotary shaker with a 2" throw for 72 hours at 28 C. At the end of the incubation period, the contents of 10 such flasks were combined and filtered with the aid of diatomaceous earth to remove the mycelium. The resulting filtered fermentation broth gave an inhibition zone of 29 mm. when assayed versus Proteus vulgarz's MB-838 by the procedure described above.

EXAMPLE 4 2.6 liters of the filtered fermentation broth prepared as described in Example 2 above and 3 liters of the filtered fermentation broth prepared in Example 3 above were combined and found to give an inhibition zone of 26 mm. when assayed with Proteus vulgaris MB-838 as described above. The 5.6 liters of fermentation broth was ad justed to pH 7.0 and passed through a column containing 100 ml. of a strongly basic anion exchange resin composed of quaternary ammonium exchange groups attached to a styrene-divinylbenzene polymer lattice (Dowex 1 x 2) on the chloride cycle at the rate of 10 ml. per minute. The resulting spent broth had no antibiotic activity when assayed with Proteus vulgarz's as described above.

The resulting resin adsorbate was washed with about 100 ml. of water and then eluted with a 3% solution of ammonium chloride in methanol. The eluate was collected in 50 ml. fractions, which exhibited the following activities by the above-described standard Proteus vulgaris assay procedure:

(1 No activity (2) 26 mm. zone (3) 28 mm. zone at 1:25 dilution (4) 26 mm. zone at 1:100 dilution (5) 35 mm. zone (6) 22 mm. zone (7) 18 mm. zone (8-10) No activities Fraction No. 4, which contained most of the antibiotic, was concentrated under vacuum to remove the methanol and then diluted with water to 50 ml. The aqueous solution of Antibiotic 833A so obtained had a pH of 5.6, a total solids of 37 mg./ml., and gave a 25.5 mm. zone at a dilution of 1:100 when assayed with Proteus vulgaris as described above.

Paper strip chromatography of this solution of Antibiotic 833A following the procedures described in EX- ample 1 show the following R.F.s:

Paper strip electrophoresis of the same product following the procedure of Example 1 showed the following:

Movement toward anode, Bloantographio organism cm.

Proteus vulgaris MB-838 l1. 8 Erwina atmseptica MB1159 10. 1

EXAMPLE 5 ml. of this aqueous solution of (cis-1,2-epoxypropyl)-phosphonic acid was chromatographed on 35 g. of 100-200 mesh spherical polyacrylamide gel having an operating range of 200-2,000 (Bio-gel P-2). The gel adsorbate was then developed with distilled water at the rate of 25 ml. per hour and 4.6 ml. eluate fractions collected. Fractions Nos. 17-21 showed antibiotic activity when assay by the Proteus vulgaris disc assay method and showed a purity of about 3 to 5 times the purity of the starting material.

During the elution with the distilled water, the column was monitored with a differential recording refractometer and salt was found in Fractions 21-24 having a K of 1.88. The K of the antibiotic active fractions was found to be 1.56.

EXAMPLE 6 A lyophilized culture of Streptomyces fradiac MA-2898 (ATCC 21096) was used to inoculate 50 ml. of sterilized medium having the following composition:

g./liter Yeast extract 10 Glucose 10 MgSO .7H O 0.5

Phosphate buffer, 2 ml. Distilled water q.s., balance.

91 g. KH POt and 95.0 g. with distilled water.

The inoculated flask was then placed on a 220 r.p.m. rotary shaker with 2 throw and incubated for 48 hours at 28 C.

An inoculum of 10 ml. of the resulting vegetative growth was then used to inoculate a 2-liter baflled Erlenmeyer flask containing 500 ml. of sterilized medium of the same composition shown above. The inoculated flask was then placed on a 220 r.p.m. rotary shaker and incubated for 48 hours at 28 C.

The resulting fermentation broth was used to inoculate a SO-gallon stainless steel fermenter containing 160 liters of sterile medium of the same composition shown above. The inoculated medium was incubated at 28 C. with agitation while maintaining an airflow of 3 c.f.m. through the fermenting broth. During the fermentation period, small amounts of Polyglycol 2,000 were added to control foaming of the batch.

An inoculum of liters of the resulting fermentation broth was then used to inoculate a 200-gallon stainless steel fermenter containing 510 liters of sterile medium of the following composition:

NnzHPOi made up to 1 liter g./liter Rolled oats 20 Distillers solubles 10 Soybean meal 20 Sodium citrate 2 Sodium ascorbate 0.5

Distilled water q.s., balance.

The pH of this medium was adjusted to 6.5 before sterilization. The inoculated broth was then incubated at a temperature of 28 C. with agitation while maintaining an airflow of 10 c.f.m. for 3 days. During the fermentation, Polyglycol 2,000, an antifoam agent, was added in small quantities to prevent excessive foaming of the fermentation broth. The fermentation broth was then filtered with the aid of diatomaceous earth. The filtered broth had a pH of 7.9 and contained 13.4 mg./ml. of total solids. Assay of the filtered broth by the Proteus vulgaris disc plate assay procedure showed the broth to contain 0.8 units/ml. of (cis-1,2-epoxypropyl)-phosphonic acid to an activity of the broth solids of 0.06 units/mg.

100 gallons of the filtered broth was adjusted to pH 7 and passed through a column containing a strongly basic anion exchange resin of the quaternary ammonium type having a styrene-divinylbenzene matrix (Dowex 1 x 2). The resulting resin ad-sorbate was eluted with a 3% solution of ammonium chloride in 90% methanol and the 24 eluate collected in 2-gallon fractions. Fractions 4,5 and 6, which contained most of the antibiotic, had the following analysis:

Total Fraction solids, Assay, Potency. Number Ing./ml units/ml units/mg.

tionation, desalting, and concentration of substances with molecular weights from 200-2,000 (available under the trade name Bio-gel P-2 from Bio-Rad Laboratories, Richmond, Calif). The resulting gel adsorbate was developed with water at the rate of 50 ml. per minute while the eluate was monitored with a refractometer. The eluate was recovered in fractions, assayed, and the appropriate active fractions combined. The 635 ml. of eluate between 5,600 ml. and 6,230 ml. was found to contain 19.1 mg./ ml. of total solids and had an activity of 68 units/ml. when assayed by the above-described Proteus vulgaris disc assay procedure. This represented (cis-1,2-epoxypropyl)-phosphonic acid having a potency of 3.6 units/ mg.

Four additional chromatograms were carried out in this same way using, respectively, 275 ml. and 287 ml. portions of the concentrate of fraction 5 and two 285 ml. portions of the concentrate of fraction 4. The rich fractions from all five of the chromatograms were pooled and concentrated under reduced pressure to about 100 mg./ml. The 660 ml. of the resulting concentrate had potency of 4.1 units/mg. v

100 ml. of this concentrate was passed through a column containing 2,480 ml. of strongly acidic cation exchange resin of the sulfonate type having a styrenedivinylbenzene matrix (sold by the Dow Chemical Company under the trade name Dowex 50 x 2) on the hydrogen form. The resulting resin adsorbate was developed with water at the rate of 19 ml. per minute and'20.5 ml. fractions of the resulting eluate collected, the eluate being monitored by a recording refractometer. Fractions 45-65 were titrated separately to pH 7 with 0.1 N sodium hydroxide. Fractions 52-62 were then assayed and pooled to obtain an aqueous solution of the antibiotic.

In the same way, five additional 100 ml. portions of the concentrate were chromatographed on the strongly acidic cation exchange resin and the most active fractions of the resulting resin eluates were combined with fractions 52-62 to obtain a total of 1,870 ml. This aqueous solution of (cis-1,2-epoxypropyl)-phosphonic acid had a pH of 6.8, a total solids content of 2.9 mg./ml. and assayed at units/ml. Thus, the potency of the sodium salt of the antibiotic in this aqueous solution was 24 units/ mg. The aqueous solution was then concentrated under reduced pressure and freeze-dried to obtain 4.7 g. of product.

The dried product so obtained exhibited the following antibacterial and cross-resistance spectrurns:

Antibacterial spectrum Test Organism: Inhibition Zone diameter, mm.

Escherichia coli MB-60 25 Bacillus sp. MB-833 7 Proteus vulgar-1's MB-l012 42 Pseudomonas aeruginosa MB-979 7 Serratia marccscens MB-252 l5 Staphylococcus (llllEIlS MB-108 17. Bacillus subtilis MB-965 29 Sarcina lutea MB-llOl 20 Staphylococcus aureus MB698 (strcptothricin- Cross-resistance spectrum Test Organism 2 Inhibition zone diameter mm.

(disc:7 mm.) 4 mg./ml.

1 (Disc:7 mm.) 4 mg./ml.

Tests performed versus a series of antibioticresistant E. coli strains isolated by exposure of the parent (MB-60) strain to inhibitory concentrations of antibiotic.

In addition to the pattern of sensitivity in the bacterial spectrum and cross-resistance assays shown above, the efiect of the addition of various materials to the medium of the E. coli control plate, which affects the activity of various antibiotics ditferently as revealed by a change in zone size, was studied. The results of these tests are shown in the following table:

SPECIAL EFFECTS SPECTRUM Inhibition zone diameter, mm. (disc=7 mm.)

Test Assay Additive 4 mg./ml.

Escherichia coli Noneeontrol 25 Do Dowexsoluble resin ET 91 24 Do 0.1 M phosphate buffer, pH 9 Do 0.1 M phosphate butler, pH 7..--.. 7

Do 0.1 M phosphate buffer, pH 9..-..- 7

Paper strip chromatography on Whatman 3 mm. filter paper dried after saturation with 0.033 M pH 7.0 phosphate butler and developed with n-butanol saturated with 0.033 M pH 7.0 phosphate buffer revealed the antibiotic to be a water-soluble compound RE:O. Paper strip electrophoresis developed for 2% hours at 600 volts with 0.165 M pH 7.0 phosphate buffer in a refrigerated apparatus revealed an unusually mobile acidic spot 11.5 cm. from origin. The presence of the activity was visualized for both the paper strip chromatography and the paper strip electrophoresis by bioautography on agar plates seeded with Escherichia coli MB60.

In summary, (cis-1,2-epoxypropyl) -phosphonic acid was shown to be a water soluble, electrophoretically mobile acidic substance with a novel antibacterial spectrum and cross-resistance pattern.

To determine the in vivo efiicacy of the antibiotic, mice were infected by the intraperitoneal route and treated with graded doses of the solid containing the sodium salt of the antibiotic prepared as described above both at the time of infection and again six hours later. Therapy was given by either the intraperitoneal or the oral route. At the completion of the test period, usually seven days after infection, the amount of antibiotic required to protect 50% of the mice (ED from this otherwise fatal infection was calculated. Uninfected mice also were treated on the same time schedule to determine whether was lethal (toxic) to the test animals." Oral to intraperitoneal therapy ratio shown in mice by. sodium (cis-l,2-epoxypropyl) phosphonate against three diiiei'ent infecting organisms i ED mg/mouse/d'ose" for pio/Lpfratio ior Sodiumsalt Tetracycline 'T odiurn Test organism r (i.p.) (p.o.)' (i.p.) (p.o) salt cycline Salmonella scholtmnelleri3010- .009 .041 .024 .756 5 31 Salmonella schottmnellerilsl t- 026 190 026 581 8 22 Staphylococcus i aurcns 2949 312 1. 18 006 187 4 31 *Therapy given by the indicated route at the time of infection and again at 6 hours after infection. Each figure represents one test only.

The following test results show the intraperitoneal end points of the same preparation of (cis-1,2-epoxypropyl)-phosphonic acid in comparison with tetracycline.

Intraperitoneal endpoints of the sodium salt of (eis-1,2-epoxypropyl)- phosphonic acid and of tetracycline in the mouse test. (Each figure represents one test only).

i.p. ED in rug/dose, 2 dose test Infecting organism Sodium salt Tetracycline 010 020 Salmonella echottmnelleri 3010 017 007 017 013 Salmonella schottmnelleri 1814 027 026 Salmonella gallinarnm 3069-.-. 377 055 Salmonella pnllorum 3198- .625 108 Salmonella typhimnrium 2637. 3. 57 200 Escher lchia colt MB1930- 294 059 Escherichia coliMB-2017 331 008 Klebsiella pneumoniae 306 4. 27 Klebsz'ella pneumoniae B- 4. 27 016 Proteus mirahllts 3201 156 068 Proteus moryanii 3202 937 031 Proteus onlgaris 1810 3. 57 188 Psendomonas aernginosa 2616.- 5. 0 201 Psendomonas aeruginosa 3210..-. 5. 0 267 Diplococcns pneumoniae I 37 5. 0 (p) I03 Staphylococcus anrcns 2949. 312 006 Streptococcus pyogenes 3009. 4. 27 011 Staphylococcus anrens 2957 302 002 *Piolongation of survival time p= .025. "Resistant to pencillin.

EXAMPLE 7 g./liter Yeast extract 10 Glucose 10 MgSO -7H O 0.05 Phosphate buffer 1 ml 2 Distilled water q.s., balance.

1 91 g. KH2PO4 and 95.0 g. NaeHPOi made up to 1 liter wilth distilled water.

The inoculated medium wasincubated at 28 C. for 24 hours with mechanical agitation while maintaining an' airflow of 10 c.f.m. through the fermenting'medium; a small quantity of Polyglycol 2,000 being added to control foaming. 215 liters of the resulting fermentationbroth was used to inoculate a 1500-gallon stainless steel fermenter' containing 1,200 gallons of sterile nutrient medium hav-- ing the following composition: I

a g. /liter Rolled oats 20 Distillers solubles 10 Soybean meal 20 Sodium citrate 2 Sodium ascorbate 0.5

Distilled water q.s., balance.

the antibiotic the medium being adjusted to pH 6.5 prior to sterilization. g 'Ihe incoula'ted broth was incubated at a temperature of 28 C. with agitation while maintaining an airflow at 55 c.f.m. for '2 days, Polyglycol 2,000 being added in small amounts to control the foaming.

..The resulting fermentation broth was filtered with the aid of diatomaceous earth. 550 gallons of the filtered broth having 'an actvitiy of 0.34 units/ml. was passed through acolumn of previously-used anion exchange resin of the quaternary ammonium type on a styrene-divinylbenzene matrix (Dowex 1 x 2) on the chloride cycle. The resulting resin adsorbate was eluted with a 3% aqueous solution of sodium chloride and the resulting eluate collected in 5- gallon fractions. The spent broth, after passing through the column, was inactive as determined by assay with Proteus vulgarz's. Fractions 5-12, which contained most of the antibiotic activity, was combined.

An additional 550 gallons of the filtered fermentation broth was passed through a column containing 40 gallons of new anion exchange resin of the same type described above and the resulting resin adsorbate was eluted with a 3% aqueous solution of sodium chloride, the eluate being collected in 5-gallon fractions. Fractions 2-11, containing most of the antibiotic activity, were combined with fractions 5-12 from the first chromatography and concentrated to about 7 gallons under diminished pressure. The resulting concentrate had a pH of 8.5 and a potency of 0.2 units/mg. as determined by the standard Proteus vulgarz's assay procedure described above.

This resin eluate concentrate was also assayed by a modified assay procedure using Proteus vulgaris as the assay organism. In this modified procedure, 5 ml. of inoculated medium is used in the petri dish and the antibiotic to be assayed is placed on a one-half inch paper disc. The dish with the test discs is then incubated for 18 hours at 37 C. and the zones of inhibition are measured. The potency is expressed as the quantity of solid per ml. producing a mm. zone of inhibition under these conditions. The resin eluate (25.85 liters) was found to contain 10.4 kg. of solids and gave a 25 mm. inhibition zone at a concentration of 2.7 mg./ ml.

To this 25.85 liters of eluate concentrate was added 15 kg. of acid-washed alumina and the mixture stirred for minutes at a pH of 5.7. The mixture was then filtered and the filtrate was found to contain 9.5 kg. of solids and to give a 25 mm. zone of inhibition at a concentration of 6.1 mg./ml. using the above-described modified assay procedure. The Antibiotic 833A adsorbed on the alumina was eluted with 25 liters of water; the alumina slurry being adjusted to pH 11.1 by the addition of concentrated ammonium hydroxide. After minutes of stirring, the alumina was filtered and the resulting eluate concentrated under reduced pressure to one liter. This concentrate contained 361 .g. of solids and gave a 25 mm. zone of inhibition at a concentration of 0.17 mg./ ml.

To 850 ml. of this rich concentrate was added 7,650 ml. of metahanol and the insolubles removed by filtration. The filtrate, .which contained practically all of the antibiotic, was passed through a column containing one kg. of acid-washed alumina. A methanol gradient containing 2. N ammonia in 75%, and 25% methanol, and finally 2 N aqueous ammonia, was then used to elute the alumina adsorbate; 4,000 ml. of each of the methanol concentrations and the same volume of aqueous ammonia being used for the elution. The eluate from the elution with 2 N ammonia-75% methanol was collected in 4 oneliter fractions and assayed. The assays indicated that these fractions contained insignificant activity and they were therefore discarded.

The eluate from the elution with the 2 N ammonia-50% methanol was likewise collected in four fractions of 1 liter each. The solid content and assay of these fractions were as follows:

Total Assay (mg/ml.

Total Assay (mg/ml.

solids per 25 mm.

Fraction (grams) inhibition zone) The eluate from the elution with the aqueous 2 N ammonia was collected in four fractions of 1 liter each. The first fraction contained a total of 2.9 g. of solids having an assay of 0.12 mg./ ml. per 25 mm. inhibition zone, and the second 4.0 g. of solids and an assay of 0.16 mg./1nl. per 25 mm. inhibition zone. The remaining two fractions contained only small amounts of antibiotic activity and were discarded.

Each of the eluate fractions obtained as described above was lyophilized. The solids from the last three fractions obtained from the elution with 50% methanol-2 N ammonia were combined and slurried three times with 500 ml. of methanol and then filtered. The methanol extracts were combined and concentrated to 500 ml. under reduced pressure. The resulting concentrate contained 7.5 g. of solids and gave a 25 mm. inhibition zone at a concentration of 0.006 mg./ml.

134 ml. of this concentrate was chromatographed on g. of acid-washed alumina using a continuous (exponential) gradient starting with 2 liters of 75% methanol-2 N ammonia at the rate of 1 mL/minute and continually maintaining the 2 liters of eluting solution at the same volume by adding 2 N aqueous ammonia. The resulting eluate was collected in 20 ml. fractions. The most active fractions 10-25 were combined and concentrated under reduced pressure to 1 ml., which was then dissolved in 12 ml. of methanol and an equal volume of N-butanol added. The methanol was evaporated under reduced pressure. The butanol insoluble material was removed. Assay of the butanol solution of the antibiotic by the modified assay procedure described in this example gave a 25 mm. inhibition zone at a concentration of .005 mg./ml.

EXAMPLE 8 A lyophilized culture of Streptomyces fradiae MA- 2913 (ATCC 21099) was used to inoculate 50 ml. of sterile medium of the following composition in a 250 ml. bafiied Erlenmeyer flask:

g./liter Ground oatmeal l0 Yeast hydrolysate 1O Phosphate buffer, 1 2 ml. MgSO .7H O 0.05

Distilled water q.s., balance.

91 g. KHQPO. and 95.0 g. NaaHPOr made up to 1 liter with distilled water.

The inoculated flask was then incubated for 24 hours at 28 C. on a rotary shaker. 10 ml. of the resulting fermentation broth was used to inoculate a second 250 ml. baflied Erlenmeyer flask containing 50 ml. of the same sterile medium. The resulting inoculated broth was incubated at 28 C. on a rotary shaker for 24 hours. The resulting fermentation broth was used to inoculate a 5- 31. aminomethane group with sodium. The sodium salt of (cis-1,2-epoxypropyl-phosphonic acid in the ml. of effluent assayed 480 units/ ml. The solution was freezedried to afford 71.6 mg. of solid assaying 67 units/mg.

EXAMPLE 12 A 25% methanol-2 N ammonia eluate obtained by the procedure described in Example 7 was freeze-dried to obtain 250 mg. of solids which gave a 25 mm. zone of inhibition at a dilution of 0.028 mg./ml. by the modified Proteus vulgaris assay procedure described in Example 7. The dried solid was dissolved in 5 ml. of methanol and 5 ml. of n-butanol added to the methanol solution. The resulting solution was filtered and the filtrate evaporated under reduced pressure to remove the methanol. The butanol solution was filtered to remove insoluble material and the filtrate containing the antibiotic was found to give a 25 mm. zone of inhibition at a dilution of 0.0065

mg./ ml.

EXAMPLE 13 The combined 50% and 25 methanol-2 N ammonia eluates, from the alumina chromatography of impure (cis-1,2-epoxypropyl)-phosphonic acid obtained following the procedures described in Example 7, were concentrated under reduced pressure to 500 ml. To this concentrate was added 1,500 ml. of methanol and the resulting solution filtered. The filtrate was evaporated under reduced pressure to obtain an aqueous solution of the antibiotic which, when assayed by the modified assay procedure with Proteus vulgaris described in Example 7, gave a 25 mm. zone of inhibition at a dilution of 0.0055 mg./ml.

To 2 ml. of the above concentrate was added ml. of methanol and the resulting solution was filtered. The filtrate was diluted with 60 ml. of n-butanol and the insolubles removed by filtration. The filtrate containing the antibiotic gave a mm. zone of inhibition at a dilution of 0.0025 mg./ml. The filtrate was evaporated to 8 ml. and the insoluble material separated by filtration. The resulting n-butanol solution of the antibiotic gave a 25 mm. zone of inhibition at a dilution of 0.0014 mg./ ml. by the modified Proteus vulgaris assay procedure.

EXAMPLE 14 To 20 ml. of an aqueous solution of the ammonium salt of (cis1,2-epoxypropyl)-phosphonic acid containing 1,020 mg. of solids and 204,000 units of activity as determined by the modified Proteus vulgaris assay described in Example 7 was added 440 mg. of calcium hydroxide; the resulting solution having a pH of 10.2. The solution was then evaporated to about /2 volume in vacuo with heating, at which point the pH was found to be 7.4. To this concentrate was added about 10 ml. of water and 100 mg. of calcium hydroxide and the resulting mixture again evaporated in vacuo to about /2 volume having a pH of 7.5. This concentrate was diluted with water to about ml. and then filtered. The clear filtrate was evaporated to 15 ml. in vacuo and the concentrate passed over a column of strongly acidic cation exchange resin (Dowex on the calcium cycle. The resin column was developed with water and 10 ml. cuts of the resulting effiuent were taken and assayed by means of the modified Proteus vulgarz's assay. Fractions 39, which contained most of the antibiotic activity, were combined and concentrated to about 8 ml. in vacuo with heating. The resulting concentrate was filtered and allowed to crystallize with stirring for about 3 hours. The crystalline calcium salt of (cis-1,2-epoxypropyl)-phosphonic acid was recovered by filtration and washed with water and then methanol before being dried in vacuo. The product so obtained weighed 240 mg. and was found to have an activity of 220 units/mg. when assayed by the modified Proteus vulgaris assay procedure. The optical rotation of the product was 5.7, [061 6, [M l2.5, [@1 -14.8. Elemental analysis after drying at 125 C.

32 for 2 hours in vacuo showed: C, 22.51%; H, 4.2%; and phosphorus, 15.3%.

A small amount of methanol was added to the filtrate and an additional 96 mg. of the crystalline calcium salt was obtained.

The aqueous solution of the ammonium salt of (cis-1,2-epoxypropyl)-phosphonic acid used as the starting material in his example was obtained by extracting an n-butanol solution of the antibiotic obtained following the procedures described in Examples 12 and 13 with 0.3 volume of water and separating the aqueous layers.

EXAMPLE 15 To 10 ml. of an aqueous solution of the ammonium salt of (cis-1,2-epoxypropyl)-phosphonic acid containing 200 mg. of solids and having a total activity of 54,000 units. said solution having been obtained following the procedure described in Example 14, was added 100 mg. of calcium hydroxide and the resulting solution diluted to 20 ml. with water and filtered. The filtrate was concentrated to about 8.5 ml. in vacuo with heating. The resulting concentrate was allowed to stand until the calcium salt of the antibiotic started to crystallize from solution, and the crystallization was allowed to continue with stirring overnight (17 hours). The crystalline product was filtered oil, washed with a small amount of 50% aqueous methanol followed by a small amount of methanol, and finally dried in vacuo at room temperature. The calcium salt so obtained weighed 111 mg. and had an activity of 252 units per mg. when assayed by the modified Proteus vugaris assay procedure described in Example 7. The elemental analysis of a sample of this calcium (cis-1,2- epoxypropyl)-phosphonate after drying at C. in vacuo (0.6% weight loss) was: C, 21.10%; H, 4.02%; 'P, 16.75%; and calcium, 21.5%. This crystalline calcium salt was found to have an optical rotation of [061 -1'6.2, [061 -13.2, [(1154 -7.4, [K1573 '6.8 and [M 6.7 when corrected for the loss in weight on drying.

EXAMPLE 16 To 118 ml. of an aqueous solution of the ammonium (cis-1,2-epoxypropyl)-phosphonate having a. solid content of 7.2 trig/ml. and an activity of 250 units/mg. (obtained by the water extraction of a butanol concentrate as described in Example 14) was added 2 grams of benzylamine dissolved in about 5 ml. of ethyl ether. and the resulting solution was concentrated to dryness in vacuo. To the residue was added about 5 ml. of methanol followed by about 5 ml. of benzene and the solution was reconcentrated and dried in vacuo. To the resulting dried partially crystalline product was added 6 ml. of ethanol and 2 ml. of benzene. The solution was then chilled and an additional 10 ml. of benzene was added. Crystals began to form, and a mixture of benzene-ethyl ether (5:1) was added. The crystals were filtered off and washed with 4 ml. of a mixture of benzene-ethyl ether (1:1) to yield 1.082 grams of product containing the monobenzylamine salt (cis-1,2-epoxypropyl)-phosphonic acid having a melting point of 139142 C. This product was taken up into 40 ml. of hot ethanol, the undissolved material was filtered off, and the resulting filtrate was concentrated to 10 ml. This solution was warmed to redissolve any precipitated material, cooled and then allowed to stand at room temperature for several hours. The crystalline benzylamine salt of (cis- 1,2-epoxypropyl)-phosphonic acid which settled out of the solution was filtered, washed with ethanol and dried to afiord 0.170 gram of the benzylamine salt having a melting point of 156160 C. When this product was recrystallized from 2.5 ml. of an ethanol-methanol mixture, 0.15 gram of the benzylamine salt of (cis-1,2- epoxpropyl)-phosphonic acid was obtained having a melting point of 153-15 8 C. Elementary analysis of the product confirms the empirical formula of C H NPo Addiliter fermenter containing 2,600 ml. of sterile nutrient medium of the following composition:

g./liter Steel cut oats 30 Corn steep liquor 10 Soybean meal 10 Water q.s., balance.

The pH was adjusted to 6.6 before sterilization.

The inoculated nutrient broth was then incubated at 28 C. for 4 days while agitating and aerating the fermentation broth with 3 liters of air per minute.

The resulting fermentation broth was then filtered to obtain 2,350 ml. of broth having a pH of 9.0 and an activity of 3.8 units/ml. as determined by the Proteus vulgaris disc assay procedure. 750 ml. of the fermentation broth were passed through a column containing 50 ml. of a strongly basic anion exchange resin of the quaternary ammonium type (Dowex 1 X 2) on the chloride cycle. The spent broth from the column contained only a small amount of antibiotic. The resulting resin adsorbate was eluted with a 1% solution of sodium chloride in water and 50 ml. fractions of the resulting eluate collected. The first six eluates contained the following amounts of antibiotic activity:

EXAMPLE 9 A lyophilized culture of Streptomyces fradiae MA- 2913 (ATCC 21099) was used to inoculate 50 ml. of sterile medium of the following composition in a 250 ml. baffled Erlenmeyer flask:

g./liter Ground oatmeal 10 Yeast hydrolysate 10 MgSO .7H O 0.05

resulting fermentation broth was used to inoculate a 5- a liter fermenter containing 3 liters of sterile nutrient broth of the following composition:

g./liter Ground oatmeal 30 Distillers solubles 10 Soybean meal 25 Sodium citrate 4 Sodium ascorbate 0.5

Water q.s., balance.

The medium was adjusted to pH 6.5 before sterilizing.

The inoculated medium was then incubated at 28 C. for 4 days while agitating and aerating the fermentation broth with 3 liters of air per minute; 3 ml. of a propylene glycol polymer having a molecular weight of about 2,000 (sold under the trade name of Polyglycol P2000 by the Dow Chemical Company) being added to prevent excessive foaming. The resulting fermentation broth had an activity of 5.9 units/nil. as determined by the standard assay using Proteus vulgar-is.

A second fermentation using this same procedure resulted in a broth having an activity of 6.75 units/ml.

The broths from the two fermentations were combined and filtered. The resulting filtered broth contained mg. of solids per ml. and at a dilution of 1 to 32 gave a mm. zone of inhibition when tested against Proteus vulgar-is using the modified assay procedure described in Example 7.

96.5 ml. of the broth was stirred for 40 minutes with 2.5 g. of acid-washed alumina. The mixture was then filtered and the filtrate was found to contain 20% of the activity. The filtered alumina adsorbate was washed and eluted with aqueous ammonia at a pH of 11.2. The eluate was evaporated to remove ammonia and was found to give a 25 mm. inhibition zone at a dilution of 0.125 mg./ ml. by the modified assay procedure.

EXAMPLE 10 A column of strongly basic anion exchange resin composed of quaternary ammonium exchange groups attached to a styrene-divinylbenzene polymer lattice (Dowex 1 x 2) on the chloride cycle (50100 mesh) 84.5 cm. x 1.4 cm. was prepared. The resin was washed with 725 ml. of 0.1 M sodium chloride to equilibrate the resin. 36 mg. of crude sodium salt of (cis-1,2-epoxypropyl)- phosphonic acid assaying 27 ,u/ mg. prepared by the process described in Example 6 was dissolved in one ml. of 0.1 M sodium chloride and applied to the top of the resin bed. The column was then developed with 0.1 M sodium chloride at a rate of one ml./minute collecting five ml. fractions. The effluent stream was monitored using a Mecco-Matic Model 2 refractorneter. Every fifth fraction was assayed against Pr Ieus vulgaris MB838 to locate the general area of bioactivity. Two bioactive peaks were observed: fractions 180 to 268 called fraction A-1 and 420 to 560 called fraction A-2. The fractions in each peak were combined and concentrated to 10 ml.

Each concentrate was percolated over a polyacrylamide gel (Bio-Gel P-2) cm. x 1.5 cm.) to separate the antibiotic from the sodium chloride. The effluent was monitored with Mecco-Matic Model 2 refractorneter. Fractions were assayed against Proteus vulgaris MB-838 to locate bioactivity. Bioactivity between 114 ml. and 250 ml. was pooled, concentrated, and freeze-dried. The Al fraction consisting of 1.4 mg. assayed 277 /mg. The A-2 fraction consisting of 3.5 mg. assayed u/mg.

EXAMPLE 11 A column of strongly basic anion exchange resin composed of quaternary ammonium exchange groups attached to a styrene-divinylbenzene polymer lattice (Dower 1 x 2) on the chloride cycle (200-400 mesh) resin 84.5 cm. x 1.4 cm. was prepared. The resin was washed with 800 ml. of tris(hydroxymethyl)aminomethane/HCI pH 8.0 (0.1 MCl-) buffer to equilibrate the resin. Three hundred five and one-half mg. of crude sodium salt of (cis-1,2-epoxypropyl)-phosphonic acid assaying 19 u/mg. was dissolved in 3 ml. of the same buffer and applied to the top of the resin bed. The column was developed with the same buffer at a rate of 1 mL/minute collecting five ml. fractions. The effluent stream was monitored using a Mecco-Matic Model 2 refractorneter. Every fifth fraction was assayed against Proteus vulgaris MB-838 to locate the general area of bioactivity. One bioactive peak was observed in fractions 115 to 185. Fractions 164 to 184 were pooled and concentrated to dryness. The residue was dissolved in water (final volume 12.0 ml.) and percolated over a polyacrylamide gel (Bio-Gel P-2) (143 cm. x 4.7 cm.) to separate the antibiotic from the buffer. The effluent was monitored with a Mecco-Matic Model 2 refractorneter. Fractions were assayed against Proteus vulgaris MB838 to locate bioactivity. Bioactivity in the fraction between 1,420 ml. and 1,680 ml. was pooled, concentrated, and freeze-dried. The resulting residue consisting of 89.6 mg. assayed 46 u/mg.

This impure antibiotic was dissolved in 5 ml. of Water and the solution passed through a column containing 2 ml. of strongly acidic cation exchange resin composed of nuclear sulfonic acid exchange groups attached to a styrene-divinylbenzene polymer lattice (Dowex 50) on the sodium cycle to replace the tris(hydroxymethyl) 33 tional monobenzylamine salt was obtained from workup the mother liquors.

EXAMPLE 17 To 40 ml. of a methanol solution containing 60 mg. of ammonium (cis-l,2-epoxypropyl)phosphonate (obtained by extracting a butanol solution of the salt with water as described in Example 14 and freeze-drying the resulting aqueous extract) was added 48 mg. of d-a-phenylethylamine. The resulting solution was evaporated under a stream of nitrogen and a small volume of methanol was added to the partially crystalline residue. After adding a small volume of acetone followed by a small volume of ether to induce further crystallization of the d-a-phenylethylamine salt of (cis-1,2-epoxypropyl)- phosphonic acid, the crystalline product was filtered oif and dried to yield product melting at 139-140 C.

EXAMPLE 18 To 50 ml. of sterile aqueous medium of the following and having a pH of 7.2 before sterilization was added 1 ml. of an inoculum prepared by suspending an agar slant of S. viridochromogenes MA-2867 (NRRL-34l4) in ml. of the same medium. The inoculated medium in a 250 ml. baflled Erlenmeyer flask was incubated for 2 days at 28 C. on a rotary shaker and 10 ml. of the resulting fermentation broth was used to inoculate 500 ml. of sterile aqueous medium having the composition shown above in a2-liter bafiled Erlenmeyer flask. This inoculated medium was then grown for 2 days at 28 C. on a rotary shaker and the resulting broth used to inoculate a SO-gallon stainless steel fermentor containing 100 liters of sterile medium of the same composition shown above. This inoculated medium was incubated at 28 C. for 40 hours with agitation while maintaining an airflow of 3 c.f.m. through the fermentation broth. During the fermentation, small amounts of Polyglycol 2000 were added to control the foaming of the fermenting medium.

-An inoculum of 43 liters of the resulting fermentation broth was then used to inoculate a ZOO-gallon stainless steel fermentor containing 510 liters of a sterile aqueous medium containing 2% tomato paste and 2% oatmeal. The inoculated broth was grown at 28 C. for 90 hours with agitation while maintaining an airflow of 10 c.f.m. through the fermenting medium. A filtered sample of the resulting fermentation broth gave a 33 mm. zone of inhibition when assayed using Proteus vulgaris MB-838 by the standard procedures described herein.

A portion of this broth was removed from the fermentor after 48 hours and filtered. The filtered broth gave a mm. zone of inhibition when assayed by the standard procedure using Proteus vulgaris MB-838. 10.5 liters of the filtered broth was adjusted to pH 8.5 with dilute sodium hydroxide and adsorbed on a column containing 210 ml. of a strongly basic anion exchange resin composed of quaternary ammonium exchange groups attached to a styrene divinylbenzene polymer lattice (Dowex 1 x 2) on the chloride cycle. The resulting resin adsorbate was washed with 300 ml. of water and eluted first with 1 liter of 3% aqueous ammonium bicarbonate solution and then with 1 liter of 3% ammonium bicarbonate in 70% methanol. The eluates were collected in 100 ml. fractions and each of the fractions assayed. The aqueous eluate accounted for 20% of the activity and the methanol eluate accounted for of the activity as determined by the standard assay using Proteus vulgaris MB-83 8. The aqueous eluate fractions were freeze-dried directly while the methanol eluate fractions were concentrated under vacuum and then freeze-dried. The freeze-dried solids were dissolved in water and re-assayed to determine the potency of the various fractions. Solutions of the first two ml. aqueous eluate fractions were combined to produce sample No. 62-2 and solutions of the first three methanol eluate fractions were combined to produce sample No. 62-3. Sample 62-2, having a total volume of 45 ml. and a total solid content of 30 mg./ml., gave a 25 mm. zone of inhibition at 1:200 dilution when assayed by the standard Proteus vulgaris assay procedure. Sample 62-3, having a total volume of 27 ml. and a total solid content of 47 ing/ml. gave a 25 mm. zone of inhibition at 1:400 dilution.

Paper strip chromatographic mobilities of (l) a Sample No. 62-2 and (2) Sample No. 622 mixed with an equal amount of sodium (cis-1,2-epoxypropyl)-phosphonate were determined by the procedure described in Example 1 with the following results:

R.F. in solvent system Biographic organism K C (1) Proteus vulgaris MB-838. 0. 42 0. 79 (2) Proteus culgarts MB-838 0. 44 0. 8

Inhibition zone diameter,

mm. (disc.=7 111111.), calcium (eis-1,2-epoxypropyl)-phosphonate Sample Test organism 400 agJml. No. 62-2 Escherichia coli MB-SO. 26 19 Bacillus sp MB833 7 7 Proteus vulgarts MB-101 39 36 Pseudomouas aerugiuosa MB-979- 8 7 Serratia marcesceus MB-252 17 13 Staphylococcus aureus MB-108. 18 12 Bacillus subttlts MB-965 36 19 25 19 Staphylococcus aureus MB 98 (Streptoth elm-resistant) 29 30 Streptococcus faecalis MB-753 12 7 Alcaligeues faecalis MB-10 11 8 Brucella bronchisepttca MB965 7 7 Salmonella gallinarum MB187 34 28 Vibrio percolaus MB-l272- 37 32 Xarzthomouas vesz'catort'a MB-81 15 7 Escherichia coli MB-GO sensitive parent 26 19 Streptomycin resistant strain 23 18 Streptothricin resistant strain. 21 12 Oxarnyein resistant strain 26 15 Pleocidin resistant strain 36 36 Chloramphenicol resistant strain 11 9 Chlortetraeycllne resistant strain 13 13 Oxytetracycline resistant strain 13 7 Neomycin resistant strain 45 49 Tetracycline resistant strain 31 20 Viomyein resistant strain. 15 10 Polymyxln resistant strai 12 19 Grisein resistant strain- 23 15 A lyophilized culture of S. viridochromogenes MA-2903 (NRRL-3413) was used to inoculate 50 m1. of a medium having the following composition:

g./ liter Yeast extract 10 Glucose 10 MgSO .7H O 0.05

Phosphate buffer, 2 m1. Distilled water q.s., to volume.

91 g. of KHzPOi plus 95.0 g. of NazHPOi made up to 1 litter in distilled water.

The inoculated medium in a 250 ml. bafiled Erlenmeyer flask was then incubated at 28 C. for 3 days on a rotary shaker. This first generation seed flask was then used to inoculate a series of like flasks for a second generation seed stage, which were shaken for 1 day in the same manner as the first generation flask. The second generation seed stage was used to inoculate (using 2.5% inoculum) a series of ten 2-liter bafilecl Erlenmeyer flasks, each containing 350 ml. of the following medium:

g./liter K HPO 0.7 KH PO 0.3 Sodium citrate.2H O 0.5 MgSO .7H O 0.1 Casamino acid 2.0 Dextrose (sterilized separately) 10 Distilled water q.s., to volume.

The resulting inoculated flasks were incubated at 28 C. for 5 days on a rotary shaker running at 145 r.p.m. with a 2-inch throw. The resulting broths were pooled, filtered and assayed. The filtered broth had a pH of 7.6 and gave a zone of inhibition of 29 mm. when assayed with Proteus vulgarz's.

EXAMPLE 20 A lyophilized culture of S. viridochromogenes MA- 2867 (NRRL-3414) was used to inoculate 50 ml. of a medium having the following composition:

Percent Casein hydrolysate (N-Z amine) 1 Dextrose 1 NaCl 0.5 Meat extract 0.3

The inoculated medium in a 250 ml. baflled Erlenmeyer flask was then incubated at 28 C. for 6-7 days on a rotary shaker. The resulting fermentation broth (2.5% inoculum), was used to inoculate ten 2-liter bafiled Erlenmeyer flasks, each containing 350 ml. of an aqueous medium of the following composition:

G./liter Casein hydrolysate (N-Z amine) 2.5 Meat extract 1.0 NaCl 5.0 Soybean meal 10 Distillers solubles 2 Corn steep liquor 5 Dextrose 2O K HPO 2 CaCo 10 Distilled water q.s., to volume.

1 Added after pH medium was adjusted to 7.0 with sodium hydroxide.

The resulting inoculated flasks were incubated for 3 days at 28 C. on a rotary shaker. At this time the fermentation broths were pooled and the filtered broth assayed using Proteus vulgarz's as the test organism. The final pH of the filtered broth was 6.6 and the broth was found to give a 30 mm. zone of inhibition.

EXAMPLE 21 A lyophilized culture of S. fradiae MA-2915 (NRRL- 3417) was used to inoculate 50 ml. of the medium described in Example 19. The 250 ml. baflled Erlenmeyer flask containing the inoculated medium was incubated at 28 C. on a rotary shaker for 1 day. The resulting fermentation broth was then used to inoculate a second seed stage using the same procedure as for the first stage. This second stage seed was used, at 2.5%, to inoculate ten 2-liter baflled Erlenmeyer flasks containing 350 ml. of a medium containing g. of dry precooked oatmeal and 20 g. of tomato paste per liter of medium. The inoculated flasks were then incubated at 28 C. for 3 days on a rotary shaker. At this time the broths were cooled and 36 assayed. The filtered broth had a pH of 6.3 and gave a 25 mm. zone of inhibition upon assay with Proteus vulgaris using the standard assay procedure.

EXAMPLE 22 A culture slant of S. viridochromogenes MA2 916 (NRRL3415) was used to inoculate 40 ml. of an aqueous medium of the following composition:

G. liter Corn starch 10 Yeast hydrolysate 10 Phosphate buffer, 2 ml. Magnesium sulfate 0.05 Distilled water q.s., to volume.

01 g. of KHQPOi plus 95.0 g. of Na2HPO4 made up to 1 liter in distilled water.

The inoculated medium in a 250 ml. baflled Erlenmeyer flask was incubated at 28 C. on a rotary shaker for 2 days. This seed was used, at 1.5%, to inoculate two 2- liter baffled Erlenmeyer flasks, each containing 350 ml. of the following medium:

G./liter Oats 20 Soybean meal 15 Sodium citrate.2H O 4.0 Distillers solubles 5 CoCl .6H O 0.1 Distilled water q.s., to volume.

The pH of this medium was adjusted to 6.5 and 0.5 of Polyglycol 2000 added to each flask individually. The resulting inoculated flasks were incubated for 4 days at 28 C. on a rotary shaker. The resulting broths were pooled, filtered and assayed. The filtered fermentation broth had a pH of 8.5 and gave a 34.5 mm. zone of inhibition upon assay with Proteus vulgaris by the standard assay procedure.

EXAMPLE 23 A culture slant of S. viridochromogenes MA-2917 (NRRL-3416) was used to inoculate 40 ml. of a medium of the following composition:

G./liter Corn starch 10 Yeast hydrolysate 10 Phosphate butler, 2 ml. MgSO 0.05 Distilled water q.s., to volume.

91 g. of KHBPO4 plus 95.0 g. of NZIZHPO4 made up to 1 liter in distilled water.

The inoculated medium was incubated in a 250 ml. baflied Erlenmeyer flask at 28 C. on a rotary shaker. The resulting fermentation broth was used, at 3%, to inoculate 22 250 ml. bafiled Erlenmeyer flasks, each containing 30 ml. of the following medium:

G./liter Oats 20 Soybean meal 15 Sodium citrate.2H O 4 Distillers solubles 5 CoCl .6H O 0.1

Distilled water q.s., to volume.

The pH of the medium was adjusted to 6.5 and 0.5% of Polyglycol 2000 was added. The inoculated flasks were grown at 28 C. for 4 days on a rotary shaker. The broths were then pooled, filtered and assayed. The filtered broth had a pH of 9.3 and gave a 25 mm. zone of inhibition upon assay at 1:8 dilution by the standard Proteus vulgaris assay.

EXAMPLE 24 A lyophilized culture of Streptomyces wedmorensis MA-3269 (NRRL-3426) was used to inoculate ml. of sterile yeast extract-glucose medium of the composition shown in Example 19 in a 250 ml. baffled Erlenmeyer flask. This negative inoculum was developed by incubation for3 days at 2 8 C. on a rotary shaker at 220 r.p.m. The resulting inoculum was used to inoculate 50 ml. of sterile casein hydrolysate-meat extract medium of the composition shown in Example in 250 ml. Erlenmeyer flasks using a 3% (1.5 nil/flask) inoculum. The inoculated flasks were grown for 4 days at 28 C. on a rotary shaker at 220 rpm. The resulting broth was centrifuged and the clarified broth was found to give a mm. zone of inhibition with 7 mm. paper discs when assayed by the standard procedure using Proteus vulgaris MB-838 as the test organism.

Additional assays on this broth using this same disc agar diffusion procedure run at 25 C. and 37 C. for 20 hours showed zones of inhibition of 7 mm. (the zone of the disc) and 26 mm. respectively. This dilference in the zones of inhibition at the two temperatures is characteristics of (cis-1,2-epoxypropyl)phosphonic acid activity.

The paper strip chromatographic mobilities determined on the clarified broth following the procedures described in Example 1 using Proteus vulgaris MB-838 as the biographic organism showed an R.F. of 0.25 in solvent system K and 0.75 in solvent system C.

EXAMPLE 25 To 50 ml. of sterile yeast extract-glucose medium of the composition shown in Example 19 in a 250 ml. Erlenmeyer flask was added 1 m1. of an inoculum prepared by suspending an agar slant of Streptomyces wedmorensis MA-32 69 (NRRL-3426) in 10 ml. of the same medium The inoculated flask was incubated for 2 days at 28 C. The resulting broth was used to inoculate 5.0 ml. of sterile casein hydrolysate-meat extract medium of the composition shown in Example 2 0 in 250 ml. Erlenmeyer flasks using a 2% inoculum. The inoculated flasks were incubated for 4 days at 28 C. on a 220 rpm. shaker. Assays of the fermented broth using the standard disc assay procedure with Proteus vulgaris MB-838 after 3 and 4 days of incubation on the centrifuged broth showed the presence of 6.2 and 6.8 units of (cis-1,2- epoxypropyl)-phosphonic acid activity respectively.

EXAMPLE 26 A portion of the inoculum prepared by incubating Streptomyces wedmorensis in the yeast extract-g1ucose medium described in Example 25 was used to inoculate (2% inoculum) 50 ml. of sterile aqueous mediums containing 2% tomato paste and 2% precooked oatmeal in 250 ml. Erlenmeyer flasks. The resulting flasks were incubated for 4 days at 28 C. on a 220 r.p.m. shaker. Assays of the fermented centrifuged broth after 3 and 4 days of incubation by the standard disc assay procedure with Proteus vulgaris MB-838 showed the presence of 3.7 and 3.5 units of (cis-1,2-epoxypropyl)phosphonic acid activity respectively. Additional assays on the clarified broth by the same assay procedure run at 25 C. and 37 C. gave zones of inhibition of 7 mm. (the zone of the 7 mm. disc) and 37 mm. respectively.

EXAMPLE 27 A solution of 5.1 g. of the phenethylamine salt of (l,2-epoxypropyl)phosphonic acid in 15 ml. of water was passed through a column of '81 ml. of a strongly acidic polystyrene-type cation exchange resin (Dowex 50X4) on the hydrogen cycle; and the column developed with water. After a fore-run of 41 ml. (1,2-epoxypropyl)phosphonic acid started to appear in the eluate, which was signalled by a drop in pH to below about 1, the next ml. of the eluate was immediately frozen and lyophilized at 25 microns pressure. The residue consisted of crystalline (cis-1,2-epoxypropyl)- phosphonic acid which melted instantly at temperatures above about 94 C. when placed in a preheated oil bath at this temperature or above. When the melting point is determined by heating the sample from room temperature, even when heated at the rate of 60/min., no melting is observed even upon heating to 170 C. The crystalline product had the following rotations at 2.5 C.: 8.7 at 578 mg, 7.84 at 546 mg, 12.6 at 436 m and 16.1 at 405 mu.

EXAMPLE 28 To 27.7 g. of mono-(+)-a-phenethylammonium (cis-1,2-epoxypropyl)phosphonate was added 87 ml. of 1.18 N sodium methoxide-methanol solution, and the mixture stirred until all of the solids dissolved. The solution was cooled to 10 C. and filtered. To this filtered solution was added 87 ml. of sodium methoxide-ethanol solution (1.15 N) (prepared by dissolving 57.87 g. of sodium methoxide in 900 ml. of anhydrous ethanol) with stirring under a nitrogen atmosphere. To this solution was added 81 m1. of absolute ethanol, and the mixture stirred at 0 C. for 30 minutes and then at room temperature for 2 hours. The mixture was then filtered and the solid disodium (cis-1,2-epoxypropyl)phosphonate was washed with 200 ml. of ethanol and dried to con stant weight at 60 C. in vacuo. A 5% solution of the disodium salt had a pH of 8.85, an

[a] i -139 and an [a] a h-51 EXAMPLE 29 A solution of 4.9 g. of the mono-benzylamine salt of (cis-1,2-epoxypropyl)phosphonic acid in 50 ml. of water was cooled to 05 C. and passed over a column containing 33 ml. of a sulfonic acid cation exchange resin of the polystyrene type (Dowex 50) on the acidic cycle at 2-4 ml. per minute; the resin having previously been cooled to O2 C. The resin column was then Washed with 225 m1. of 05 C. Water at the same rate. The effluents were collected directly in an agitated solution of 1.50 g. of sodium hydroxide in 10 m1. of Water and the pH of the resulting solution was then adjusted to pH 8.7 by the addition of dilute sodium hydroxide. This solution was concentrated in vacuo at 25 C. or less to a net weight of about 1520 g. and to this largely solidified product was added 30 ml. of methanol with stirring. To the resulting slurry is added ml. of isopropanol and this mixture was concentrated to about 75 ml. in vacuo at 25 C. The volume was then readjusted to .125 ml. with isopropanol, stirred for 15 minutes and filtered. The solid filter cake consisting of disodium (cis-1,2-epoxypropyl)-phosphonate was dried at 60 C. in vacuo owernight. A 5% solution of the disodium salt in water had a pH of 8.5 and an gigg -lrz When the foregoing process was repeated using an equivalent amount of potassium hydroxide in place of the sodium hydroxide dipotassium (cis-l,2-epoxypropyl)-phosphonate was obtained. A 5% aquous solution of this product in water had a pH of 8.8-9.0 and an EXAMPLE 30 To 3.6 g. of disodium salt and 4.28 g. of dipotassium salt was added suflicient water to dissolve the salts. The resulting solution was concentrated to dryness in vacuo at 25 C. to obtain the monosodium monopotassium mixed salt of (cis-1,2-epoxypropyl)phosphonic acid. A 5% solution of this mixed salt had a pH of 8.89.0 and an EXAMPLE s1 Mono-uphenethylammonium (cis-1,2-epoxypropyl)-phosphonate monohydrate, 27.7 g. (0.1 mole),

39 was dissolved in 450 ml. of water. This solution was stirred with magnesium oxide, 8.0 g. (0.2 mole), for 3 hours at 5 C. The excess magnesium oxide was filtered off and the filtrate concentrated in vacuo to 100 grams. The concentrate was diluted with 100 ml. of methanol and 800 m1. of isopropanol was slowly added. The precipitate is filtered and the cake washed with 200 ml. of isopropanol and dried in vacuo to give 14.5 g. of magnesium (cis-1,2-epoxypropyl)-phosphonate dihydrate having [04%; 13 (c.=%, water) EXAMPLE 32 A cooled (0-5 C.) solution of 32 g. of mono-(+)-(a)- phenethylammonium (cis-1,2-epoxypropyl) phosphonate in 480 ml. of Water was passed over 200 ml. of a sulfonic acid cation exchange resin of the polystyrenetype (Dowex 50) on the hydrogen cycle at a flow rate of about 15 ml. per minute. The effiuent and the succeeding 400 ml. of cold water-wash was collected in a solution of 4.6 g. of sodium hydroxide in 50 ml. of water. The pH of the resulting solution was 5.4 and was adjusted to 7.0 with 6 N sodium hydroxide solution. The resulting solu tion was reduced in vacuo at 25 C. to a volume of about 75 ml. This concentrate was then filtered to obtain a solution of sesqui sodium (cis-1,2-epoxypropyl)- phosphonate. Evaporation of the solution in vacuo at a temperature of C. afforded the sesqui sodium salt as a white solid. A 2% solution of this product in water has a pH of 6.8 and an EXAMPLE 33 Following the procedures of Example 32 but using an equivalent amount of the appropriate amine, the following amine salts were obtained mono-(+ )-amphetammonium (cis-1,2-epoxypropyl)phosphonate, melting at 163.5165 C.

monoethylammonium (cis-1,2-epoxypropyl)-phosphonate, melting at 152153 C.

monodiethyl-ammonium (cis-l,2-epoxypropyl)- phosphonate, melting at 120.5122.5 C.

mono-6-threo-1-phenyl-1,3-dihydroxy-2-propylammonium (cis-1,2-epoxypropyl)-phosphate, melting at 103105 C.

ethylenediamine salt of (cis-1,2-epoxypropyl)- phosphonate, melting at 98100 C.

mono-piperizine salt of (cisl,2-epoxypropyl)- phosphonic acid, melting at 188 C.

diethylenetriamine salt of (cis-1,2-ep0xypropyl)- phosphonic acid, melting at 177-179" C.

hexamethylene-l,6-diamine salt of (cis'1,2-epoxypropyl)-phosphonic acid, melting at 194 C.

N,N'-dibenzylethylenediamine salt of (cis-1,2- epoxypropy1)-phosphonic acid, melting at 151- 152 C.

bis-guanidine salt of (cis-1,2-epoxypropyl)-phosphonic acid, melting at 235260 C.

ethylenediamine-N,N'-bis (cis-1,2-epoxypropyl)- phosphonate, melting at 175210 C.

mono-ethyl glycinate salt of (cis-l,2-epoxypropyl)- phosphonic acid.

mono-L-(+)-lysine (cis-1,2-epoxypropyl)-phosphonate, melting at 165195 C.

mono- )brucine (cis-1,2-epoxypropyl phosphonate, melting at 210-220 C.

mono-procaine (cis-1,2-epoxypropy1)-phosphonate,

melting at 12l125 C.

EXAMPLE 34 To a solution of 5.27 g. of mono-(+)-o-phenethylammonium (cis-l,2-epoxypropyl)-phosphonate monohydrate in 27 ml. of water was added about 1.16 ml. of

50% sodium hydroxide to a final pH of about 9.0. The

40 resulting solution was filtered and to this solution was added a filtered solution of 3.35 g. of calcium acetate monohydrate dissolved in 13 ml. of water at a temperature below about 30 C. The resultingfwhite slurry was aged about minutes and then filtered. The wet cake was washed with water to remove the phenethylamine. The, resulting calcium (cis-1,2-epoxypropyl)-phosphonate monohydrate was air-dried and had an at concentration of 5% in aqueous ethylenediamine tetraacetic acid at pH 8.8.

EXAMPLE 35 A cooled (0-5 C.) of 11.08 g. of mono-(+)-u-phenethylammonium (cis-1,2-epoxypropy1)-phosphonate in 160 ml. of water was passed over 120 ml. of a sulfonic acid cation exchange resin of the polystyrene-type (Dowex on the hydrogen cycle at a fiow rate of about 5 ml. per minute. The effluent and the succeeding 125 ml. of cold water wash was collected and 7.5 g. of aluminum nitrate monohydrate was added. The pH of the solution was then adjusted to 6.0 with 50% sodium hydroxide, and then evaporated to about 32.3 g. in vacuo at 25 C. To this concentrate was added 58 ml. of ethanol and the solution was stirred for an hour. 25 ml. of ethanol was added and the solution was filtered. The solid aluminum salt of (cis-1,2-epoxypropyl)-phosphonic acid was washed with ethanol and dried at 60 C. in vacuo.

What is claimed is:

1. An antibacterial composition comprising an effective antibacterial amount of (cis-1,2epoxypropyl)phosphonic acid or a pharmaceutically acceptable salt thereof and a carrier.

2. A process for the production of (cis1,2-epoxypropyl)phosphonic acid which comprises growing a (cis-l,2-epoxypropyl)phosphonic acid producing Streptomyces from the group consisting of Streptomyces fradiae NRRL B3357, B-3358, B-3359, B-3360 or 3417, Streptomyces virzdochromogenes NRRL 3413, 3414, 3415, 3416 or 3427 and Srreptomyces wedmorensis NRRL 3426 in an aqueous nutrient medium under aerobic conditions until substantial antibiotic activity is imparted to said medium.

3. A process according to claim 2 wherein the Streptomyces is a strain of Streptomyces fradiae NRRL B-3357, 13-3358, B3359, B-3360 or 3417.

4. A process according to claim 2 wherein the Streptomyces is a strain of Streptomyces viridochromogenes NRRL 3413, 3414, 3415, 3416 or 3427.

5. A process according to claim 2 wherein the Streptomyces is a strain of Streptomyces wedmorensis NRRL 3426.

6. A process for the production of (cis-l,2-epoxypropyl)phosphonic acid which comprises growing a (cis-1,2-epoxypropyl)phosphonic acid producing strain of Streptomyces from the group consisting of Streptomyces fradiae NRRL B-3357, B-3358, B-3359, B3360 or 3417, Streptomyces virz'dochromogenes NRRL 3413, 3414, 3415, 3416 or 3427 and Streptomyces wedmorensis NRRL 3426 in an aqueous nutrient medium under aerobic conditions until substantial antibiotic activity is imparted to said medium, and recovering (cis-1,2-epoxypr0pyl) phosphonic acid from the resulting fermentation broth.

7. A process according to claim 6 wherein the Streptomyces is Streptomyces fradiae NRRL B3357, B-3358', B3359, B4360 or 3417. 1

8. A process according to claim 6 wherein the Streptomyces is Streptomyccs viridochromogenes NRRL 3413, 3414, 3415, 3416 or 3427.

9. A process according to claim 6 wherein the Streptomyces is Streptomyces weclmorcnsis NRRL 3426.

(References on following page) 

